Cinnamomi and poria composition, method to prepare same and uses thereof

ABSTRACT

This invention provides a method for identifying a composition of Cinnamomi and Poria. This invention further provides a composition comprising: Ramulus Cinnamomi, Poria Cortex, Moutan Radicis, Radix Paeonize Alba, and Semen Persicae. This invention provides various uses of these compositions.

This application is a Continuation of U.S. Ser. No. 11/389,441, filedMar. 24, 2006, which is a Continuation of U.S. Ser. No. 10/403,187,filed Mar. 31, 2003, now U.S. Pat. No. 7,052,700, which is aContinuation of U.S. Ser. No. 09/951,070, filed Sep. 13, 2001, now U.S.Pat. No. 6,569,468, which is a Continuation-In-Part application ofInternational Application No. PCT/CN00/00273, filed Sep. 13, 2000, thecontents of which are incorporated herein into this application byreference.

Throughout this application, various publications are referenced andfull citations for these publications may be found in the references atthe end of the specifications preceding the claims. The disclosures ofthese publications are hereby incorporated by reference into thisapplication in order to more fully describe the state of the art asknown to the skilled therein as of the date of the invention describedand claimed herein.

BACKGROUND OF THE INVENTION

Menstrual cramps and dysfunctional uterine bleeding are common disordersamong women during their menstrual period. Menstrual cramps affect asmany as 40 percent of female adults and temporarily disables one-tenthof them. As a newly developed treatment, the present invention—Cinnamomiand Poria composition, which originated from an old Chinese herbformula, is effective in relieving symptoms of such diseases ordisorders.

Menstrual cramps, or dysmenorrhea, as it is called in medical terms, isthe reoccurrence of painful uterine cramps during menstruation.Dysmenorrhea can include crampy pain; discomfort in the lower abdomen,lower back and the inner thighs; nausea; vomiting; headache; diarrhea;or fatigue. It normally has an onset of from 2-12 hours before the startof menses and tapers over the next one to two days.

Primary dysmenorrhea accounts for a majority of the cases in which nopathologic lesions can be found in pelvic or laparoscopic examination.Although the exact etiology is unspecified, recent advances inbiochemistry of prostaglandins to study chemicals and their role inpathophysiology have now established a rational basis for some cases ofdysmenorrhea. The release of prostaglandins is significantly increasedduring menstruation in these women, and the release can be suppressed tonormal levels when non-steroidal anti-flammatory drugs (NSAIDs) aregiven, since NSAIDs are capable of inhibiting cyclo-oxygenase andsynthesis of prostaglandins.

In addition to NSAIDs, oral contraceptives, antispasmodics, andanalgesics are commonly used by physicians. Androgen therapy issometimes used, and a minor surgery called dilation and curettage (D&C)is also adopted in certain cases.

Traditional treatments are associated with a variety of side effects,including ineffectiveness and drug tolerance, and have limited effects.That explains why primary dysmenorrhea is still a problem. The presentinvention was developed using the most common herbal formulations inTraditional Chinese Medicine (TCM). This invention uses a differentapproaching characterized by steady effectiveness and relatively lowtoxicity.

The Cinnamomi and Poria composition is synthesized to protect women fromthe pain caused by most common pelvic diseases or disorders. In additionto treating primary and secondary dysmenorrhea and dysfunctional uterinebleeding caused by irregular shedding of uterine endometrium, thecomposition is also effective in treating chronic pelvic inflammations,inflammatory lower abdominal masses and small intramural hysteromyoma.

Like most TCM products, the Cinnamomi and Poria composition is preparedfrom multiple medicinal herbal materials—five cultivated natural plantscalled Ramulus Cinnamomi, Poria, Cortex Moutan, Radix Paeoniae Alba andSemen Persicae. Different in weights and portions of the medicinal herbsfor the start-up materials and in the producing courses, Cinnamomi andPoria composition shares the same formula of five herbs with a previousmedicinal preparation—Bolus of Cinnamomi and Poria (BCP).

BCP, which has long been approved as an effective cure, was a sample ofsuccess in medicinal practice in ancient China. The formula of five wassourced from Jinkui Summary, a guide compiled by famous TCM practitionerZhongjing Zhang, some 1,000 years ago. It was also collected in manymedicine directory resources, such as the “Traditional Chinese MedicineDictionary.”

With different courses of preparation, the five-plant formula was alwaysadministered in combination with other medicinal herbs. It claims totreat a variety of diseases and disorders, including many indications inthe present invention.

As a comparison, while BCP was prepared by the extraction of herbs'water solution—a more traditional way in TCM practice, the presentinvention is produced by updated technologies which could betterpreserve its effective ingredients. Cinnamomi and Poria composition wasdeveloped in 1989, and have obtained the Certificate of New MedicineProduct Permit by authorities in China.

In regards to the medicinal herb materials and their ingredientsinvolving in present invention, a variety of United States Patents andliteratures that taught methods in composing curative compositions fromherbs like Cinnamomi, Poria etc. A variety of reports from China,country of the inventor, are also cited which were mostly in connectionwith animal or clinical researches for BCP and certain embodiment ofCinnamomi and Poria composition.

In U.S. Pat. No. 6,093,403, the formula consist an extract of Poriacomposition up 5 to 20% in weight are been used as a cure or preventionof disorders in sugar balance, diabetes, and such blood circulationdiseases such as Angina Pectoris.

Poria cocos wolf is being use for treating diseases occurred in area ofcardio, cerebro-vascular, and for that of Alzheimer's and depression. InU.S. Pat. No. 5,589,182 contain Poria cocos wolf up to 20% of its weightmake up.

During the menopause stage for many women, many of them might experiencea period, which is called “Hot Flashes”. In U.S. Pat. No. 5,874,0874,which is a herbal formula for treating women, who are in the stage ofHot Flashes. This herbal formula contains both Peony Root and PoriaCocos, making up to 13.3% of the weight of the formula.

U.S. Pat. No. 5,942,233, discloses a composition that consists of semenPersicae (Peach Kernel) up 30% of its weight. This formula is known forusages on blood stimulation and the flow of vital energy.

A pharmaceutical liquid composition which contains Cinnamomi Cortex isdisclosed in U.S. Pat. No. 5,225,203 which amount 66 parts and 56 partsin Paeoniae Radix of its weight. This pharmaceutical liquid was used inhospital everywhere for treating patients that are suffering fromstroke, arteriosclerosis, hypertension, tachycardia, dyspnea, anxiety,cardiostenosis, acute and chronic convulsion, automatic nervous systemdisease, and coma.

Another pharmaceutical liquid composition containing Paeoniae Radix andCinnamomi Cortex are allegedly being used in U.S. Pat. No. 5,133,964,which presents an invention that relates to the preparation of oral andparental natural substance liquids of improve on physical stability. Inthis formula, it contains about 66 mg of Cinnamomi Cortex and 56 mg ofPaeoniae Radix. The formula is also being used for the same treatmentsas described in U.S. Pat. No. 5,225,203.

U.S. Pat. No. 4,613,591 describes an adminiculum for antitumor agents.The composition consists both of Cinnamomi Cortex and Paeoniae Radixfrom 2.0 to 4.0 parts of the formula. The composition is used as acontrol substance for side effects and adminiculum for mitomycin C anddoxorubicin.

Xie, J Z et al. published an article in 1986 on BCP's influences onblood flow of rabbits. Xie says that BCP had long been claimed for itseffect against hypostasis. In their test on rabbits both with oral andintravenous administration, Xie observed that the whole blood reducedspecific viscosity and plasma specific viscosity were lowered, RBCelectrophoresis time shortened and fibrinogen reduced.

Tu, Z K S et al. in 1988 reported their research results or oraladministration of BCP in 10 health human bodies on their blood flow,blood platelet functions etc. The results showed that the whole bloodviscosity was significantly lowered after two weeks of administration,the blood platelet coagulation induced by collagen and ADP and synthesisof thromboxane B₂ in blood platelets was also inhibited. But, thediagrams of thrombus elasticity showed no changes.

Xie, J Z et al. reported BCP's effect on central nerve system in 1987.Xie reported that oral and injection of BCP in mice both lead to asignificant analgesia and sedation effect to the animals. Xie furtherpointed out that the half lethal dose was 80.0±10.9 g/kg if giventhrough subcutaneous injection, and over 250 g/kg if given orally.

He, H Q et al. published an article in 1994 on clinical study ofCinnamomi and Poria composition in 100 cases of gynecopathies:dysfunctional uterine bleeding caused by irregular shedding of uterineendometrim, chronic pelvic inflammations, dysmenorrhea and smallintramural hysteromyoma. In comparison with 50 cases of patients thatwere treated with BCP, observations over multiple complaining symptomsand physical signs had showed no significant difference between the twogroups.

Shi, D M et al. reported in 2000 after observing of therapeutic effectsof Cinnamomi and Poria composition in 60 patients with hysteromyoma.Patients accepted physical examination on gynecology, ultrasonicexamination and hematochrome test. The effectiveness of treatment wasdefined as significant improvement: tumor reduced 3-5 cm and enlargedmenstrual flow reduced 50% or over; improvement: tumor reduced 2-3 cmand menstrual flow reduced 25% or over and ineffective: underimprovement. The results showed in Shi's report that an effective rateof 91.7% was acquired, and among which, 10% of the cases was ofsignificant improvement.

In one embodiment of the invention, Capsule of Cinnamomi and Poria (CCP)was made. And pre-clinical studies on CCP's pharmacological effects haveshowed that it has the effects of uterine smooth muscle relaxation,antispasmodic, pain-releasing and anti-inflammation, best explanationfor its indication of dysmenorrhea and chronic pelvic inflammations.

Cinnamomi and Poria composition with different concentrations may causean inhibitory effect against the contraction of extra-corporeal uterineof rats. It counteracts the acceleration and enhancement of contractionsof the extra-corporeal uterine that was induced by oxytocin. Cinnamomiand Poria composition solution of different concentrations might resultin the decrease of blood platelet agglutination rate in rats. Theeffectiveness had a positive relation with different concentrations.Cinnamomi and Poria composition could also induce a downfall of bloodviscosity in rats administered with it for a few days.

Reaction to pains caused by chemical or heat, mice will twist theirbodies or swing their tails. By recording the times of body twisting ortail swinging, an objective index of their feeling of pains could beevaluated. CCP, if given to the animals a few days before the tests,will, within certain scope, relieve them from the body movement.

For the effect of anti-inflammation, it could be observed that withadministration of CCP, the weights of mice's swelling ears were reducedobviously. The weight of mice's swelling ear reflects the severity ofinflammation in the ear that was smeared with croton oil at its surfacea period of time before.

A general study of pharmacology on rats' cardiovascular, respiration andnerve system showed that CCP had no noticeable reverse effectphysiologically on the above systems. The acute toxicity test wasconducted on 20 mice with 246 g/kg in dosage (a thousand times ofhuman's clinical dosage), administered by gastric injection. No death orany other abnormal activities were found. The 90 days long-term toxicitystudy was also negative in general health indexes, general bloodperimeters, functions and pathological studies of different organs.

The controlled clinical studies of multi diseases and comprehensiveevaluations were conducted among women of China. 150 people wereinvolved in the first stage trial (Phase II). No deaths or seriousadverse events had been reported. The clinical studies showed positiveresults in a comprehensive evaluated analysis: the evaluation to amultiple indications: dysmenorrhea, chronic pelvic inflammations andmany others.

SUMMARY OF THE INVENTION

This invention provides a method for identifying a composition ofCinnamomi and Poria comprising the steps of: a) dissolving thecomposition of Cinnamomi and Poria with an appropriate aqueous solutionmixed with an appropriate organic solvent; and b) performing gaschromatographic analysis using a HP-5 5% phenyl methyl siloxanecapillary column.

This invention also provides a method for identifying a composition ofCinnamomi and Poria comprising the steps of: a) dissolving thecomposition of Cinnamomi and Poria with an aqueous solution; and b)separating the dissolved mater with an C 18 column under high pressureliquid chromatography.

This invention furthermore provides a method for identifying acomposition of Cinnamomi and Poria comprising: extracting thecomposition of Cinnamomi and Poria by an appropriate organic solvent;using appropriate standard for liposoluble matter in the composition ofCinnamomi and Poria, as internal control; and performing high pressureliquid chromatography using a C18 column.

Furthermore, this invention provides a composition comprising:

a) 1.3-1.9% paeoniflorin and

b) 0.7-1.1% Paeonol.

This invention provides a method for obtaining a composition ofCinnamomi and Poria comprising steps of: a) obtaining, pruning, washingand cutting the plant parts: stem of Cinnamomum cassia Presl (Fam.Lauraceae), fungus of Poria cocos (Schw.) Wolf (Fam. Polyporaceae), Rootof Paeonia suffruticosa Andr. (Fam. Ranunculaceae). and fruit of Prunuspersica (L.) Batsch or Prunus davidiana (Carr.) Franch. (Fam. Rosaceae);b) drying the said plants to form 5 medicinal materials: RamulusCinnamomi, Poria Cortex, Moutan Radicis, Radix Paeonize Alba and SemenPersicae; c) Smashing Ramulus Cinnamomi, Semen Persicae and MoutanRadicis into coarse powders and chopping Radix Paeonize Alba into slice;d) Sterilizing the Poria Cortex before granulating 50% of its formulaweight into fine powder and filtering the powder; e) putting full amountof powder of Cortex Moutan through a process of hot reflux in water andcollecting its distillate; (residue and fluid reserved); f) filteringand vacuum drying said distillate to obtain crude Paeonol; g) dissolvingthe crude paeonol into 95% alcohol; Slowly adding the solution intosaturated water solution of β-cyclodextrin while agitating it atthermostatic 80° C. to form a mixture A; h) exhausted filtering mixtureA; washing the residue with anhydrous alcohol and letting it dried, aclathrate A is obtained; i) distilling full amount of Ramulus Cinnamomiin water for four hours and collecting its volatile matter; (residue andfluid reserved) j) dissolving the volatile matter into 95% alcohol;slowly adding the solution into saturated water solution ofβ-cyclodextrin while agitating it at thermostatic 45° C. to form amixture B; k) exhausted filtering mixture B; washing the residue withanhydrous alcohol and letting it dried, a clathrate B is obtained; l)mixing residues from step (f) and (j) with full amount of Radix PaeonizeAlba, Semen Persicae, 50% of Poria Cortex and 90% alcohol; extractingthe mixture, filtering the extract and recovering alcohol from thefiltered extract; (residue reserved) m) adding water in residue fromstep (m), distilling it and filtering the water extract; n) mixing waterextract from step (n), alcohol extract from step (m), fluid from step(f) and step (j); enriching the mixture to form a creamed extractive; o)mixing the creamed extractive with Poria Cortex powder from step (e);grinding the mixture into fine powder after vacuum drying it to form agranule; p)

-   -   mixing the fine powder with some 60% alcohol and starch gum;        Granulating the powder to 30 meshes; q) mixing certain amount of        silicon dioxide with clathrate A from step (i) and clathrate B        from step (l); and r) mixing the mixture from step (r) with the        granule from step (p) to obtain a final granule—the composition        of Cinnamomi and Poria.

Furthermore, this invention provides a pharmaceutical composition fortreating gynecological blood stasis, cardio-cerebral vascular diseases,respiratory system and urinary system diseases comprising the followingmaterials in weight proportion: Ramulus Cinnamomi, 1-2 portion; PoriaCortex, 1-2 portion; Moutan Radicis, 1-2 portion; Radix Paeonize Alba,1-2 portion; and Semen Persicae, 1-2 portion.

Finally, this invention also provides a method for obtaining acomposition comprising: distilling a required amount of Cortex Moutanwith water through a process of hot reflux and collecting itsdistillate, filtering and drying said distillate after its cooling downto obtain crude Paeonol; mixing the residues with required amount ofRamulus Cinnamomi, Radix Paeonize Alba, Semen Persicae and 50% of PoriaCortex, adding alcohol to the mixture and extract, extracting themixture, filtering the extract to obtain an alcohol extract; addingwater to residue from the alcohol extraction, extracting and filteringit to obtain a water extract; Mixing the alcohol extract, water extractand solution from distilling of Cortex Moutan, enriching the mixture toa creamed extract; and granulating the rest 50% of Poria Cortex intofine powder, mixing the fine powder with the creamed extract,granulating the mixture after vacuum drying, mixing the granule withcrude Paeonol; and filling the mixture in to capsules to form theproduct.

DETAILED DESCRIPTION OF THE FIGURES

FIG. 1. Gas Chromatography of CCP's volatile matter

-   -   Preparation of the volatile matter: Add 50 ml water and ml ethyl        ether to the content of 10 capsules of Cinnamomi and Poria.        Extract the solution with reflex on a Water bath of 75° C. for        90 minutes and let it cool down. Extract the water layer of the        solution with 20 ml ethyl ether for three times and merge all        ethyl ether solutions together. Volatilize to dry up the ethyl        ether solution on a water bath of 35° C. and, finally, add ethyl        ether to make it 5 ml in volume.    -   System Requirement: HP-5 5% phenyl methyl siloxane capillary        columns (30.0 m×0.32 mm×0.25 um); columns temperature 80° C. (5        min) to 250° C. (10 min) rising at 3° C./min; carrier gas is        nitrogen with flow rate 1.5 ml/min; FDI detector with hydrogen        40 ml/min, air 350 ml/min; makeup gas: nitrogen 30 ml/min; inlet        system temperature 250° C.; split injection with 50:1 in split        ratio and 2 μl. in volume; temperature of detector 280° C.;        recording time 72 minutes.

FIG. 2. HPLC of CCP's Water-soluble matter

-   -   Preparation of the water-soluble matter: Add 200 ml water to the        content of 3 capsules. Extract the solution with reflex for 30        minutes (after boiling) and let it cool down. Centrifuge the        solution for 10 minutes and filtrate its supernatant with 0.45        um filter membrane to obtain the filtrate.    -   System Requirements: Spectrum column: Alltima C₁₈ 5 um, 7.5        mm×4.6 mm (guard column) and 250 nm×4.6 mm (spectrum column);        column temperature 30° C.; flow rate 1 ml/min, detective        wavelength 230 nm; injection volume 5 ul; recording time 70        minutes.

FIG. 3. HPLC of CCP's liposoluble matter in 210 nm of detectivewavelength

-   -   Preparation of the Liposoluble Matter: Use the precipitation        deposit that obtained from after the centrifugal process in        water-soluble solution preparation. Flush the deposit with water        until it becomes colorless. Add 20 ml methanol to the deposit,        extract the solution with reflex for 30 minutes (after boiling)        and let it cool down. Centrifuge the solution for 10 minutes.        Volatilize to dry up the methanol from the solution on a water        bath of 75° C. And finally, add certain amount of methanol to        make the solution to be at 2 ml in volume, filtrate it with 0.45        um filter membrane and obtain the filtrate.    -   System Requirements: Spectrum column: Alltima C₁₈ 5 um, 7.5        mm×4.6 mm (guard column) and 250 nm×4.6 mm (spectrum column);        column temperature 50° C.; flow rate 1 ml/min, detective        wavelength 210 nm and 242 nm; injection volume 5 ul; recording        time 76 minutes.

FIG. 4. HPLC of CCP's liposoluble matter in 242 nm of detectivewavelength

-   -   Preparation of Sample Solution of Liposoluble Matter: same with        method of FIG. 3.    -   System Requirements: same with method of FIG. 3.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a method for identifying a composition ofCinnamomi and Poria comprising the steps of: a) dissolving thecomposition of Cinnamomi and Poria with an appropriate aqueous solutionmixed with an appropriate organic solvent; and b) performing gaschromatographic analysis using a HP-5 5% phenyl methyl siloxanecapillary column.

This invention also provides a method for identifying a composition ofCinnamomi and Poria comprising the steps of: a) dissolving thecomposition of Cinnamomi and Poria with an aqueous solution; and b)separating the dissolved mater with an C 18 column under high pressureliquid chromatography.

This invention furthermore provides a method for identifying acomposition of Cinnamomi and Poria comprising: extracting thecomposition of Cinnamomi and Poria by an appropriate organic solvent;using appropriate standard for liposoluble matter in the composition ofCinnamomi and Poria, as internal control; and performing high pressureliquid chromatography using a C18 column.

In an embodiment, this invention provides a composition of Cinnamomi andPoria comprising the product when subjected to the above method produces7 peaks as shown in FIG. 1. In another embodiment, this inventionprovides a composition of Cinnamomi and Poria comprising the productthat when subjected to above method produces 13 peaks as shown in FIG.2. In another embodiment, this invention provides a compositioncomprising the product when subjected to the above method produces 7peaks at 210 nm of detective wavelength as shown in FIG. 3. In yetanother embodiment, this invention provides a composition comprising theproduct when subjected to the above method produces 8 peaks at 242 nm ofdetective wavelength as shown in FIG. 4.

In a separate embodiment, the invention provides a compositioncomprising the product when subjected to the above method produces afingerprint as tabulated herein:

Retention Range of Peak Number Time/Area Retention Time Range of Aria 10.757/1.183 0.770~0.745 1.740~0.690 Standard 1/1 2 1.275 1.280~1.270 31.290/0.696 1.295~1.285 1.110~0.350 4 1.449/5.565 1.455~1.4408.080~3.460 5 1.704/0.093 1.710~1.700 0.140~0.060 6 2.146/0.1642.150~2.140 0.255~0.080 7 3.061/0.115 3.070~3.055 0.160~0.070

In another embodiment, this invention provides a composition comprisingthe product that when subjected to the above method produces afingerprint as tabulated herein:

Retention Range of Peak Number Time/Area Retention Time Range of Aria 10.261/0.645 0.275~0.250 0.750~0.460 2 0.349/0.103 0.360~0.3400.160~0.070 3 0.584/0.128 0.600~0.560 0.230~0.065 4 0.915/0.2120.920~0.910 0.250~0.170 Standard 1/1 5 1.076/0.089 1.085~1.0700.130~0.065 6 1.118/0.046 1.125~1.110 0.060~0.035 7 1.162/0.0521.175~1.155 0.080~0.030 8 1.196/0.083 1.210~1.180 0.105~0.055 91.268/0.076 1.285~1.250 0.090~0.065 10 1.312/0.211 1.330~1.2950.255~0.140 11 1.420/0.404 1.450~1.400 0.470~0.310 12 2.107/0.1492.170~2.060 0.195~0.130 13 2.389/0.981 2.465~2.340 1.475~0.680

In an embodiment, this invention provides a composition comprising theproduct that when subjected to the above method at 210 nm of detectivewavelength produces a fingerprint as tabulated herein:

Retention Range of Peak Number Time/Area Retention Time Range of Aria 10.367/0.322 0.375~0.355 0.540~0.180 2 0.408/0.580 0.420~0.3950.900~0.410 3 0.897/0.280 0.905~0.890 0.350~0.220 4 0.980/0.7520.985~0.975 0.940~0.600 Standard 1/1 5 1.019/0.286 1.025~1.0150.410~0.210 6 1.143/4.650 1.150~1.135 5.950~2.900 7 1.305/0.9591.315~1.295 1.450~0.575

In still another embodiment, this invention provides a compositioncomprising the product that when subjected to the above method at 242 nmof detective wavelength produces a fingerprint as tabulated herein:

Retention Range of Peak Number Time/Area Retention Time Range of Aria 10.520/0.155 0.530~0.510 0.185~0.120 2 0.566/0.184 0.570~0.5600.230~0.140 3 0.686/1.385 0.690~0.680 1.950~0.900 Standard 1/1 41.128/0.481 1.135~1.125 0.530~0.430 5 1.557/0.181 1.585~1.5250.240~0.150 6 1.763/1.414 1.800~1.720 1.700~1.250 7 2.133/0.6302.190~2.070 0.840~0.480 8 3.033/0.463 3.105~2.950 0.680~0.160

In another embodiment, the invention provides the above composition,wherein: the retention time (RT) ratios of the 7 peaks in comparisonwith cinnamaldehyde are 0.757, 1.275, 1.290, 1.449, 1.704, 2.146 and3.061 respectively; the area ratios of peak 1 and peaks 3-7 incomparison with cinnamaldehyde are 1.183, 0.696, 5.565, 0.093, 0.164,0.115 respectively; and the range of the retention time ratios of the 7peaks in comparison with cinnamaldehyde are 0.770-0.745, 1.280-1.270,1.295-1.285, 1.455-1.440, 1.710-1.700, 2.150-2.140 and 3.070-3.055respectively; and the range of the area ratios of peak 1 and peaks 3-7in comparison with cinnamaldehyde are 1.740˜0.690, 1.110˜0.350,8.080˜3.460, 0.140˜0.060, 0.255˜0.080 and 0.160˜0.070 respectively.

This invention also provides the above composition, wherein: theretention time (RT) ratios of the 13 peaks in comparison withpaeoniflorin are 0.261, 0.349, 0.584, 0.915, 1.076, 1.118, 1.162, 1.196,1.268, 1.312, 1.420, 2.107 and 2.389 respectively; the area ratios ofthe 13 peaks in comparison with paeoniflorin are 0.645, 0.103, 0.128,0.212, 0.089, 0.046, 0.052, 0.083, 0.076, 0.211, 0.404, 0.149 and 0.981respectively; and the range of the retention time ratios of the 13 peaksin comparison with paeoniflorin are 0.275-0.250, 0.360-0.340,0.600-0.560, 0.920-0.910, 1.085-1.070, 1.125-1.110, 1.175-1.155,1.210-1.180, 1.285-1.250, 1.330-1.295, 1.450-1.400, 2.170-2.060 and2.465-2.340 respectively; and the range of the area ratios of the 13peaks in comparison with paeoniflorin are 0.750-0.460, 0.160-0.070,0.230-0.065, 0.250-0.170, 0.130-0.065, 0.060-0.035, 0.080-0.030,0.105-0.055, 0.090-0.065, 0.255-0.140, 0.470-0.310, 0.195-0.130 and1.475-0.680 respectively.

Furthermore, the invention provides the composition above, wherein: theretention time (RT) ratios of the 7 peaks at 210 nm of detectivewavelength in comparison with pachymic acid are 0.367, 0.408, 0.897,0.980, 1.019, 1.143 and 1.305 respectively; the area ratios of the 7peaks in comparison with pachymic acid are 0.322, 0.580, 0.280, 0.752,0.286, 4.650 and 0.959 respectively; and the range of the retention timeratios of the 7 peaks in comparison with pachymic acid are 0.375-0.355,0.420-0.395, 0.905-0.890, 0.985-0.975, 1.025-1.015, 1.150-1.135 and1.315-1.295 respectively; and the range of the area ratios of the 7peaks in comparison with pachymic acid are 0.540-0.180, 0.900-0.410,0.350-0.220, 0.940-0.600, 0.410-0.210, 5.950-2.900 and 1.450-0.575respectively.

This invention also provides the above composition, wherein: theretention time (RT) ratios of the 8 peaks at 242 nm of detectivewavelength in comparison with polyporenic acid C are 0.520, 0.566,0.686, 1.128, 1.557, 1.763, 2.133 and 3.033 respectively; the arearatios of the 8 peaks in comparison with polyporenic acid C are 0.155,0.184, 1.385, 0.481, 0.181, 1.414, 0.630, and 0.463 respectively; andthe range of the retention time ratios of the 8 peaks in comparison withpolyporenic acid C are 0.530-0.510, 0.570-0.560, 0.690-0.680,1.135-1.125, 1.585-1.525, 1.800-1.720, 2.190-2.070 and 3.105-2.950respectively; and the range of the area ratios of the 8 peaks incomparison with polyporenic acid C are 0.185-0.120, 0.230-0.140,1.950-0.900, 0.530-0.430, 0.240-0.150, 1.700-1.250, 0.840-0.480,0.680-0.160 respectively.

Furthermore, this invention provides a composition comprising:

a) 1.3-1.9% paeoniflorin and

b) 0.7-1.1% Paeonol.

In an embodiment, the invention provides the above composition extractedfrom Ramulus Cinnamomi, Poria, Cortex Moutan, Radix Paeoniae Alba andSemen Persicae.

In a separate embodiment, this invention provides the above composition,wherein the Ramulus Cinnamomi, Poria, Cortex Moutan, Radix Paeoniae Albaand Semen Persicae are obtained from cultivated plants.

This invention provides a method for obtaining a composition ofCinnamomi and Poria comprising steps of: a) obtaining, pruning, washingand cutting the plant parts: stem of Cinnamomum cassia Presl (Fam.Lauraceae), fungus of Poria cocos (Schw.) Wolf (Fam. Polyporaceae), Rootof Paeonia suffruticosa Andr. (Fam. Ranunculaceae). and fruit of Prunuspersica (L.) Batsch or Prunus davidiana (Carr.) Franch. (Fam. Rosaceae);b) drying the said plants to form 5 medicinal materials: RamulusCinnamomi, Poria Cortex, Moutan Radicis, Radix Paeonize Alba and SemenPersicae; c) Smashing Ramulus Cinnamomi, Semen Persicae and MoutanRadicis into coarse powders and chopping Radix Paeonize Alba into slice;d) Sterilizing the Poria Cortex before granulating 50% of its formulaweight into fine powder and filtering the powder; e) putting full amountof powder of Cortex Moutan through a process of hot reflux in water andcollecting its distillate; (residue and fluid reserved); f) filteringand vacuum drying said distillate to obtain crude Paeonol; g) dissolvingthe crude paeonol into 95% alcohol; Slowly adding the solution intosaturated water solution of β-cyclodextrin while agitating it atthermostatic 80° C. to form a mixture A; h) exhausted filtering mixtureA; washing the residue with anhydrous alcohol and letting it dried, aclathrate A is obtained; i) distilling full amount of Ramulus Cinnamomiin water for four hours and collecting its volatile matter; (residue andfluid reserved) j) dissolving the volatile matter into 95% alcohol;slowly adding the solution into saturated water solution ofβ-cyclodextrin while agitating it at thermostatic 45° C. to form amixture B; k) exhausted filtering mixture B; washing the residue withanhydrous alcohol and letting it dried, a clathrate B is obtained; l)mixing residues from step (f) and (j) with full amount of Radix PaeonizeAlba, Semen Persicae, 50% of Poria Cortex and 90% alcohol; extractingthe mixture, filtering the extract and recovering alcohol from thefiltered extract; (residue reserved) m) adding water in residue fromstep (m), distilling it and filtering the water extract; n) mixing waterextract from step (n), alcohol extract from step (m), fluid from step(f) and step (j); enriching the mixture to form a creamed extractive; o)mixing the creamed extractive with Poria Cortex powder from step (e);grinding the mixture into fine powder after vacuum drying it to form agranule; p) mixing the fine powder with some 60% alcohol and starch gum;Granulating the powder to 30 meshes; q) mixing certain amount of silicondioxide with clathrate A from step (i) and clathrate B from step (l);and r) mixing the mixture from step (r) with the granule from step (p)to obtain a final granule—the composition of Cinnamomi and Poria.

In an embodiment, the invention provides a method for obtaining acomposition of Cinnamomi and Poria composition above further comprisingthe steps of: I. in step (e) filtering the powder with a 100 meshessift; in step (f) soaking Cortex Moutan in water for four hours beforedistillation; in step (g) vacuum drying the distillate at below 55° C.and the concentration of crude paeonol should be no less than 80%; instep (i) and (l) continuously agitating the mixtures at thermostatic 80°C. (mixture A) and 45° C. (mixture B) for another 3 hours and storing itfor 24 hours in a refrigerator before it is filtered; drying theclathrates to less than 2% moisture; in step (j) soaking RamulusCinnamomi in water for six hours before distillation and testing thepresence of cinnamaldehyde in the volatile matter; in step (m)extracting the mixture twice for two hours each in 3 times weight ofresidues and medicinal materials of 90% alcohol; in step (n) distillingthe residue from step (m) twice for two hours each in 4 times weight ofthe residues of water; in step (o) enriching the mixture in vacuum attemperature below 55° C. to a relative density of no less than 1.27(75-80° C.); in step (p) vacuum drying the powder at below 55° C.; instep (q) vacuum drying the powder at below 55° C.; in step (r) mixingthe clathrates with silicon dioxide in a high performance mixer; andmass balance in formula and above-mentioned respective steps should beat ±5%.

In a separate embodiment, the creamed extractive from step (o) is noless than 1.27 in relative density; component of paeoniflorin is1.8%-2.7%; and limit of heavy metal is 5 ppm. In another embodiment, thefine powder from step (p) is no more than 5.0% in moisture; component ofpaeoniflorin is 1.4%-2.2%; and limit of heavy metal is 5 ppm. In yetanother embodiment, the clathrates is no more than 2.0% in moisture;Paeonol and cinnamaldehyde tests are positive; and limit of heavy metalis 5 ppm. In yet another embodiment, the final granule from step (s) isno more than 4.0% in moisture; cinnamaldehyde tests is positive;component of paeoniflorin is 1.3%-1.9%; component of Paeonol is0.7%-1.1%; and limit of heavy metal is 10 ppm.

This invention further provides the composition comprising the productproduced by the methods above.

In addition, this invention provides a method for determination of totalpaeoniflorin and total paeonol in a Cinnamomi and Poria compositioncomprising steps of: a) preparing the assay comprising the steps of:accurately weighing a suitable amount of standard paeoniflorin;dissolving it in chromatographically pure methanol to form a standardsolution containing 0.9 mg. of standard paeoniflorin per 1 ml; dilutingsaid standard solution of paeoniflorin to 18.0 μg./ml standard solutionbefore using; accurately weighing a suitable amount of standard paeonol;dissolving it in chromatographically pure methanol to form a standardsolution containing 0.3 mg. of standard paeonol per 1 ml; diluting saidstandard solution of paeonol to 6.0 μg./ml standard solution beforeusing; accurately weighing approximately 0.5 g. of granular of Cinnamomiand Poria composition; and dissolving it with 20 ml. water to form awater solution; putting said solution in ultrasound for 10 minutes fordispersing; extracting the solution in a separating funnel with 30 ml.analytical pure chloroform each time for 5 times; obtaining a solution Aby combining the extracts; distilling solution A on a water bath of 70°C. to a suitable volume; mixing it with analytically pure chloroform toa constant volume of 50 ml in a measuring flask; VII. accurately sucking5 ml. of the 50 ml. solution from step (VI); mixing it with analyticallypure chloroform to a constant volume of 50 ml; and filtrating thediluted solution with 0.45 μm. filtration membrane to obtain a samplesolution I; extracting the water solution left from step (V) withanalytical pure water saturated n-butanol 30 ml. each time for 5 times;combining the extracts; mixing the combination from step (VIII) withanalytical pure water saturated n-butanol to a constant volume of 200 mlin a measuring flask to obtain solution B; distilling to dry 50 mlsolution B on a water bath; dissolving the residue withchromatographically pure methanol to a constant volume of 25 ml in ameasuring flask; and accurately sucking 5 ml. of the 25 ml. solutionfrom step (IX); mixing it with chromatographically pure methanol to aconstant volume of 25 ml; filtrating the diluted solution with 0.45 μm.filtration membrane to obtain sample solution II. b) using methanolsolution of paeoniflorin (18.0 μg/ml) and methanol solution of paeonol(6.0 μg/ml) obtained from steps (II) and (IV) as the standard solutions;and c) performing PLC assay under following conditions: spectrum column:ALLtech-426 C₁₈ 5 μm., 4.6×250 mm; detector: ALLtech UVIS-201; pump:ALLtech-426 HPLC pump; injection valve: 7725i with 10 μl volume; columntemperature: room temperature; flow rate 1 ml/min; injection volume: 10μl; detective wavelength: 230 nm (paeoniflorin) and 274 (paeonol);mobile phase: methanol-water 35:65 (paeoniflorin) 60:40 (paeonol); andnumber of theoretical plates: based on peak of paeoniflorin, the numberof theoretical plates should be no less than 5000; base on peak ofpaeonol, the number of theoretical plates should be no less than 1500.d) performing HPLC assay with following steps: injecting 10 μl. samplesolution I (for paeonol) or sample solution II (for paeoniflorin) intoHPLC to perform chromatography under given circumstance; and measuringarea of the peaks to calculate the relative components of paeoniflorinand paeonol in the Cinnamomi and Poria composition.

In an embodiment, paeoniflorin is used as the standard to determine theamount of paeoniflorin in a Cinnamomi and Poria composition.

In separate embodiment, paeonol is used as the standard to determine theamount of paeonol in a Cinnamomi and Poria composition.

This invention also provides a pharmaceutical composition comprising aneffective amount of the composition above and a pharmaceuticallyacceptable carrier.

In an embodiment, the above formulation is a pill, capsule, granule,tablet, suspension, injection, syrup, or tincture.

In an embodiment, the invention provides a method for relaxing thesmooth muscles of excessively contracting uterine in a subject bydirectly inhibiting the contraction frequency, range and overallactivity of the smooth muscles of uterine comprising administering tothe subject an effective amount of the pharmaceutical composition above.In another embodiment, this invention provides a method for reducing thewhole blood viscosity in a subject by inhibiting the agglutination ofthe blood platelet and inhibiting the release of blood plateletcomprising administering to the subject an effective amount of thepharmaceutical composition above.

In yet another embodiment, the invention provides a method forregulating inflammatory functions in a subject by inhibiting theinflammatory reactions of various kinds comprising administering to thesubject an effective amount of the pharmaceutical composition above. Instill another embodiment, the invention provides a method for inducing aanalgesic effects onto pains of various kinds in a subject comprisingadministering to the subject an effective amount of the pharmaceuticalcomposition above. In still another embodiment, the invention provides amethod for treating primary and secondary dysmenorrhea of various kindsof degrees in a subject comprising administering to the subject aneffective amount of the pharmaceutical composition above. In anotherembodiment, this invention provides a method for alleviating clinicalsymptoms in a subject suffering from primary and secondary dysmenorrheaof various kinds comprising administering to the subject an effectiveamount of the pharmaceutical composition above.

In another embodiment, the invention provides a method for treatingdysfunctional uterine bleeding caused by irregular shedding of uterineendometrium in a subject comprising administering to the subject aneffective amount of the pharmaceutical composition above.

In yet another embodiment, the invention provides a method foralleviating clinical symptoms in a subject suffering from dysfunctionaluterine bleeding caused by irregular shedding of uterine endometriumcomprising administering to the subject an effective amount of thepharmaceutical composition above. In still another embodiment, theinvention provides a method for treating chronic pelvic inflammationsand inflammatory lower abdomen masses in a subject comprisingadministering to the subject an effective amount of the pharmaceuticalcomposition above.

In a further embodiment, the invention provides a method for alleviatingclinical symptoms in a subject suffering from chronic pelvicinflammations and inflammatory lower abdomen masses comprisingadministering to the subject an effective amount of the pharmaceuticalcomposition above.

In still another embodiment, this invention provides a method fortreating small intramural hysteromyoma in a subject comprisingadministering to the subject an effective amount of the pharmaceuticalcomposition above. In yet another embodiment, the invention provides amethod for alleviating clinical symptoms in a subject suffering fromsmall intramural hysteromyoma comprising administering to the subject aneffective amount of the pharmaceutical composition above.

In a further embodiment, the invention provides a method for alleviatingthe symptoms of Profuse or prolonged menstruation, excess of blood clotsduring menstrual period, vague pain, distending pain at lower abdomenand lower lumber, large amount of leulorrhea, heavy and distending anusor anemia in a subject suffering from dysfunctional uterine bleeding,primary and secondary dysmenorrhea, chronic pelvic inflammations,inflammatory lower abdomen masses or small intramural hysteromyomacomprising administering to the subject an effective amount of thepharmaceutical composition above.

In another embodiment, the invention provides a method for improvingabnormal indexes in blood rheology in a subject comprising administeringto the subject an effective amount of the pharmaceutical compositionabove. In another embodiment, the invention provides a method fordecreasing dim purpuric spots in tongue in a subject comprisingadministering to the subject an effective amount of the pharmaceuticalcomposition above.

In another embodiment, the invention provides a method for improvingfaint wrist pulse in a subject comprising administering to the subjectan effective amount of the pharmaceutical composition above.

Furthermore, this invention provides a pharmaceutical composition fortreating gynecological blood stasis, cardio-cerebral vascular diseases,respiratory system and urinary system diseases comprising the followingmaterials in weight proportion: Ramulus Cinnamomi, 1-2 portion; PoriaCortex, 1-2 portion; Moutan Radicis, 1-2 portion; Radix Paeonize Alba,1-2 portion; and Semen Persicae, 1-2 portion.

This invention also provides a method for obtaining a compositioncomprising: distilling a required amount of Cortex Moutan with waterthrough a process of hot reflux and collecting its distillate, filteringand drying said distillate after its cooling down to obtain crudePaeonol; mixing the residues with required amount of Ramulus Cinnamomi,Radix Paeonize Alba, Semen Persicae and 50% of Poria Cortex, addingalcohol to the mixture and extract, extracting the mixture, filteringthe extract to obtain an alcohol extract; adding water to residue fromthe alcohol extraction, extracting and filtering it to obtain a waterextract; Mixing the alcohol extract, water extract and solution fromdistilling of Cortex Moutan, enriching the mixture to a creamed extract;and granulating the rest 50% of Poria Cortex into fine powder, mixingthe fine powder with the creamed extract, granulating the mixture aftervacuum drying, mixing the granule with crude Paeonol; and filling themixture in to capsules to form the product.

In an embodiment, the composition is featured as 3 times weight of 90%alcohol for the alcohol added in the mixture and 4 times weight of waterfor the water added in the mixture. In another embodiment, the alcoholextraction is 2 hours each time and the water extraction is 2 hours eachtime.

In a further embodiment, this invention provides a compositioncomprising the product produced by the method above for treatingdiseases of gynecological blood stasis.

In yet another embodiment, the method of producing drug for treatingdiseases of gynecological blood stasis is the method of producing drugfor treating hysteromyoma. In another embodiment, the method ofproducing drug for treating diseases of gynecological blood stasis isthe method of producing drug for treating pelvic inflammations. In yetanother embodiment, the method of producing drug for treating diseasesof gynecological blood stasis is the method of producing drug fortreating dysmenorrhea. In a further embodiment, the method of producingdrug for treating diseases of gynecological blood stasis is the methodof producing drug for treating irregular menstruation. In a furtherembodiment, the method of producing drug for treating diseases ofgynecological blood stasis is the method of producing drug for treatingbleeding diseases of women.

The invention also provides the composition comprising the productproduced by the method above for treating diseases of cardio-cerebralvascular diseases. In an embodiment, the method of producing drug fortreating cardio-cerebral vascular diseases is the method of producingdrug for treating hypertension. In another embodiment, the method ofproducing drug for treating cardio-cerebral vascular diseases is themethod of producing drug for treating heart diseases.

This invention also provides the product produced by the method abovefor treating diseases of respiratory system diseases. The inventionstill further provides the composition comprising the product producedby the method above for treating diseases of urinary system diseases.

This invention provides a composition comprising 1.3-1.9%cinnamaldehyde.

This invention provides a composition comprising 0.7-1.1% paeoniflorin.

This invention provide a composition that when the volatile matter ofthe composition subjected to Gas Chromatography (GC) the 7 peakfingerprint shown in FIG. 1 and tabulated in Table 2 is produced. Inthis fingerprint the retention time (RT) ratios of the 7 peaks incomparison with cinnamaldehyde are 0.757, 1.275, 1.290, 1.449, 1.704,2.146 and 3.061 respectively; the area ratios of peak 1 and peaks 3-7 incomparison with cinnamaldehyde are 1.183, 0.696, 5.565, 0.093, 0.164,0.115 respectively; and the range of the retention time ratios of the 7peaks in comparison with cinnamaldehyde are 0.770-0.745, 1.280-1.270,1.295-1.285, 1.455-1.440, 1.710-1.700, 2.150-2.140 and 3.070-3.055respectively; and the range of the area ratios of peak 1 and peaks 3-7in comparison with cinnamaldehyde are 1.740-0.690, 1.110-0.350,8.080-3.460, 0.140-0.060, 0.255-0.080 and 0.160-0.070 respectively.

This invention provide a composition that when the Water-soluble matterof the composition subjected to High Performance Liquid Chromatography(HPLC) the 13 peak fingerprint shown in FIG. 2 and tabulated in Table 3is produced. In this fingerprint the retention time (RT) ratios of the13 peaks in comparison with paeoniflorin are 0.261, 0.349, 0.584, 0.915,1.076, 1.118, 1.162, 1.196, 1.268, 1.312, 1.420, 2.107 and 2.389respectively; the area ratios of the 13 peaks in comparison withpaeoniflorin are 0.645, 0.103, 0.128, 0.212, 0.089, 0.046, 0.052, 0.083,0.076, 0.211, 0.404, 0.149 and 0.981 respectively; and the range of theretention time ratios of the 13 peaks in comparison with paeoniflorinare 0.275-0.250, 0.360-0.340, 0.600-0.560, 0.920-0.910, 1.085-1.070,1.125-1.110, 1.175-1.155, 1.210-1.180, 1.285-1.250, 1.330-1.295,1.450-1.400, 2.170-2.060 and 2.465-2.340 respectively; and the range ofthe area ratios of the 13 peaks in comparison with paeoniflorin are0.750-0.460, 0.160-0.070, 0.230-0.065, 0.250-0.170, 0.130-0.065,0.060-0.035, 0.080-0.030, 0.105-0.055, 0.090-0.065, 0.255-0.140,0.470-0.310, 0.195-0.130 and 1.475-0.680 respectively.

This invention provide a composition that when the liposoluble matter ofthe composition subjected to High Performance Liquid Chromatography(HPLC) of 210 nm in detective wavelength the 7 peak fingerprint shown inFIG. 3 and tabulated in Table 4 is produced. In this fingerprint theretention time (RT) ratios of the 7 peaks in comparison with pachymicacid are 0.367, 0.408, 0.897, 0.980, 1.019, 1.143 and 1.305respectively; the area ratios of the 7 peaks in comparison with pachymicacid are 0.322, 0.580, 0.280, 0.752, 0.286, 4.650 and 0.959respectively; and the range of the retention time ratios of the 7 peaksin comparison with pachymic acid are 0.375-0.355, 0.420-0.395,0.905-0.890, 0.985-0.975, 1.025-1.015, 1.150-1.135 and 1.315-1.295respectively; and the range of the area ratios of the 7 peaks incomparison with pachymic acid are 0.540-0.180, 0.900-0.410, 0.350-0.220,0.940-0.600, 0.410-0.210, 5.950-2.900 and 1.450-0.575 respectively.

This invention provide a composition that when the liposoluble matter ofthe composition subjected to High Performance Liquid Chromatography(HPLC) of 242 nm in detective wavelength the 8 peak fingerprint shown inFIG. 4 and tabulated in Table 5 is produced. In this fingerprint theretention time (RT) ratios of the 8 peaks in comparison with polyporenicacid C are 0.520, 0.566, 0.686, 1.128, 1.557, 1.763, 2.133 and 3.033respectively; the area ratios of the 8 peaks in comparison withpolyporenic acid C are 0.155, 0.184, 1.385, 0.481, 0.181, 1.414, 0.630and 0.463 respectively; and the range of the retention time ratios ofthe 8 peaks in comparison with polyporenic acid C are 0.530-0.510,0.570-0.560, 0.690-0.680, 1.135-1.125, 1.585-1.525, 1.800-1.720,2.190-2.070 and 3.105-2.950 respectively; and the range of the arearatios of the 8 peaks in comparison with polyporenic acid C are0.185-0.120, 0.230-0.140, 1.950-0.900, 0.530-0.430, 0.240-0.150,1.700-1.250, 0.840-0.480, 0.680-0.160 respectively.

This invention provides the above compositions having componentsextracted from Ramulus Cinnamomi, Poria, Cortex Moutan, Radix PaeoniaeAlba and Semen Persicae.

This invention provides the above compositions having componentsextracted from Ramulus Cinnamomi, Poria, Cortex Moutan, Radix PaeoniaeAlba and Semen Persicae that are obtained from cultivated plants.

Capsule of Cinnamomi & Poria (CCP)

The product is produced in capsule form. Its interior is yellowish browngranule, witch is fragrant and tasted slightly bitter. Each capsulecontains 0.31 g. of the granule, which can be stored for 18 month inroom temperature.

CCP is used for primary or secondary dysmenorrhea, dysfunctional uterinebleeding caused by irregular shedding of endometrim, chronic pelvicinflammations with inflammatory lower abdomen masses or small intramuralhysteromyoma, and many common pelvic disorders.

CCP is for oral administration, 3 capsules each time, 3 times a day,preferably after meals. The users are advised not to use this productduring a menstruation period. Period of treatment course is three monthsor follow directions of a physician.

CCP was source from Bolus of Cinnamomi & Poria. The inventors starteddeveloping it in 1989, and had been granted the Certificate of NewMedicine and Certificate of production by authorities in China. (1995,Health Dept approval No. Z-25; Health Dept Medical Standard 1998;WS2-097 [X-017]-98 [Z])

Formula of the Raw Medicinal Materials

Ramulus Cinnamomi 144 k.g. Poria Cortex 144 k.g. Moutan Radicis 144 k.g.Radix Paeonize Alba 144 k.g. Semen Persicae 144 k.g. (For 600,000capsules)Detailed Process

Wash Ramulus Cinnamomi, Moutan Radicis, Radix Paeonize Alba and SemenPersicae in high-pressure water after picking out impurities. Fumigatingsterilize the four medicinal materials with Epoxyethane. Smash RamulusCinnamomi, Semen Persicae and Moutan Radicis into coarse powders andchop Radix Paeonize Alba into slice of 2-3 mm

Microwaves sterilize the Poria Cortex. Granulate 50% of Poria Cortex ofthe formula weight into fine powder (70-72 k.g. for the formula of totalraw materials of 720 k.g.). Filter the powder with a 100 meshes sift.

Soak full amount of Cortex Moutan (144 k.g.) in water for four hoursbefore distilling it in water and collecting its distillate. Aftercooling down of the distillate, filtering and vacuum drying it at below55° C. 1.6-1.8 k.g. crude Paeonol is obtained from the distillate. Theconcentration of crude paeonol should be no less than 80%. The residueand fluid from the above procedure is reserved for future use; Weight10.2 k.g. of β-cyclodextrin to prepare its saturated water solution at80° C. Dissolve full amount of above-mentioned crude paeonol into 9.0k.g. of 95% alcohol. Slowly add the alcoholic solution into theβ-cyclodextrin water solution while keep agitating the mixture atthermostatic 80° C. Keep agitating the mixture at thermostatic 80° C.for another 3 hours and stored it for 24 hours in a refrigerator beforeit is exhausted filtering. The residue is then washed by small amount ofanhydrous alcohol and let dried in the room temperature. About 9.86 k.g.of clathrate of white powder is obtained from this step.

Soak full amount of Cortex Moutan (144 k.g.) in water for six hours.Distill Ramulus Cinnamomi in water for four hours and collecting itsvolatile matter. (presence of cinnamaldehyde is tested here in thevolatile matter). 0.82-0.93 liter of volatile oil is obtained from thisstep. The residue and fluid is reserved for future use; Weight 3.72 k.g.of β-cyclodextrin to prepare its saturated water solution at 45° C.Dissolve full amount of above-mentioned cinnamomi volatile oil into 5.0k.g. of 95% alcohol. Slowly add the alcoholic solution into theβ-cyclodextrin water solution while keep agitating the mixing solutionat thermostatic of 45° C. Keep agitating it at thermostatic 45° C. foranother 3 hours and stored 24 hours in a refrigerator before exhaustedfiltered. The residue is then washed by small amount of petroleum etherand let dry in the room temperature. About 3.70 k.g. of white powderclathrate is obtained.

The residue of Cortex Moutan and Ramulus Cinnamomi from theabove-mentioned steps are mixed with full amount of Radix Paeonize Alba(144 kg), Semen Persicae (144 kg), the rest 50% of Poria Cortex (72 kg)and three times weight of 90% alcohol. Extract the mixture twice for twohours each, filter the extract and reserve the alcohol extract afterrecovery of the alcohol from it. Add four times weight of water to itsresidue, distill it twice for two hours each time and filter the waterextract. The water extract, alcohol extract, fluid of Cortex Moutan andfluid of Ramulus Cinnamomi from the above-mentioned steps are joinedtogether and were enriched in vacuum at temperature below 55° C. About110-130 k.g. of a creamed extractive with a relative density of no lessthan 1.27 (75-80° C.) is obtained.

Thoroughly mix the creamed extractive with the granulated Poria Cortexpowder and grind it into fine powder after a vacuum drying procedure atunder 55° C. About 164 k.g. of a powder is obtained.

Thoroughly mix 8.2 k.g. of starch gum with the fine powder. Granulatethe mixed powder to 30 meshes with some 60% alcohol. After vacuum dryingprocess at a temperature under 55° C. and rectifying procedure, a total171-172 k.g. of granule is obtained.

Thoroughly mix the β-cyclodextrin clathrate of Paeonol, β-cyclodextrinclathrate of Cinnamomi volatile oil and 0.33 k.g. of silicon dioxide ina high performance mixer. 13.8 k.g. of mixture is obtained.

Thoroughly mix the 13.8 k.g. of mixture with the above-mentionedgranule, rectify the granule. 186 k.g. of intermediate granule isproduced. Mass balance of all above-processed materials should be ±5%.

Technical Requirements

(1) The Poria fine powder needs to be screened by 100 meshes of screen(inner diameter 150±6.6 μm.)

(2) the concentration of crude paeonol should be no less than 80%;

(3) Concentration and drying must be performed under vacuumedconditions. The temperature can't be above 55° C.

(4) The volatile matter is tested for the presence of cinnamaldehyde.

(5) Material mass balance should be ±5%.

Medicinal Materials and their Processing

Ramulus Cinnamomi (Common Name: Cassia Twig)

Cassia Twig is the dried young stem of Cinnamomum cassia Presl (Fam.Lauraceae). The plant is collected in spring or summer. It is dried inthe sun after collection, removal of its leaves and perhaps chopped intoslides.

The medicinal preparation has cylindrical body, multi-branched, 30-75 cmin length, its thick end is 0.3-1 cm in diameter. It is brown orreddish-brown on the surface, with longitudinal lines, fine wrinkles,dotted with leaf, branch or bud scars, lenticels dotted or dottedelliptic. Hard and fragile, it is easily broken. For the slices, it is2-4 mm thick, cut surface showing reddish-brown in bark, yellowish-whiteto pale yellowish-brown in the wood part, pith subsquare. Odorcharacteristic aromatic; taste, sweet and slightly pungent, especiallyin bark. The medicinal preparation is the clean scraps of Cinnamomiwithout visible impurities. The important ingredients of Cinnamomi iscinnamaldehyde and cinamic acid.

Provider: Tianma Medicinal Material Company in Bozhou, Anhui P.R.C.Attn: ZHOU, Fang Mobile: 011-86-1360-5682-623

Poria Cortex (Common Name: Indian Bread)

Poria is the dried sclerotium of the fungus, Poria cocos (Schw.) Wolf(Fam. Polyporaceae). The plant is collected from July to September.After collection, it was piled up and spread about for air-dryingrepeatedly until wrinkles appears on the surface and its inner moistureevaporated. The whole dried sclerotium is known as “Fulingge”. If thefresh sclerotium is peeled before drying. The separated parts are called“Fulingpi” (peel) and “Fulingkuai” (flesh).

Fulingge Subglobose, ellipsoid, oblate or irregular shaped, variable insize. The outer skin thin and rough, brown to blackish brown,conspicuously shriveled and striated. Texture hard and compact, fracturegranular, some cracked, the outer layer pale brown, inner part white,rarely reddish, some showing the penetrating roots of pine in thecentre. Odourless; taste, weak and sticky when chewed. The medicinalpreparation is the clean scraps of Poria without visible impurities. Itsimportant active ingredients are Pachman and Pachymic acid.

Provider: Shuanggou Medicine Company in Bozhou, Anhui P.R.C. Attn:HUANG, Jigang Telephone: 011-86-558-5116-893, Mobile:011-86-1380-5689-093.

Cortex Moutan (Common Name: Tree Peony Bark)

Tree Peony Bark is the dried root bark of Paeonia suffruticosa Andr.(Fam. Ranunculaceae). The root is collected in autumn, removed fromrootlets, the root bark is stripped off, and dried in the sun.

Quilled or semiquilled, longitudinally fissured, somewhat involute oropened, 5-20 cm long, 5-12 mm in diameter, 1-4 mm thick. The outersurface greyish-brown or yellowish-brown, showing numerous transverselenticels and rootlet scars, the exposed layer where cork fallen offappearing pink; the inner surface greyish-yellow or brownish, withobvious fine longitudinal striations, usually showing bright crystals.Texture hard and fragile, easily broken, fracture relatively even,starchy, pale pink. Odour, aromatic; taste, slightly bitter andastringent. The medicinal preparation should be the clean Moutan Radiciswithout visible impurities. The important ingredients: Paeonol.

Provider: Nature Medicinal Herbs Company in Bozhou, Anhui P.R.C. Attn:ZHOU, Wenxin Telephone: 011-86-562-6811-050.

Radix Paeoniae Alba (Common Name: White Peony Root)

White Peony Root is the dried root of Paeonia lactiflora Pall. (Fam.Ranunculaceae). The drug is collected in summer and autumn, washed inwater, remove the root stock and the lower part and rootlet, boiled inwater, peeled, and dried in the sun.

Cylindrical, straight or slightly curved, two ends truncate, 5 18 cmlong, 1-2.5 cm in diameter. Externally whitish or pale reddish brown,glossy or with longitudinal wrinkles, rootlet scars and occasionalremains of brown cork. Texture compact, easily broken, fracturerelatively even, whitish or pale brownish-red, cambium ring distinct andrays radial. Odour, slight; taste, slightly bitter and sour. Medicinalused Radix Paeonize Alba should be the clean scraps of Radix Paeonizewithout visible impurities. Its important active ingredient ispaeoniflorin.

Provider: National Medicinal Herb Company in Bozhou, Anhui P.R.C. Attn:ZHOU, Hai Mobile phone: 011-86-1370-5680-735.

Semen Persicae (Common Name: Peach Seed)

Peach Seed is the dried ripe seed of Prunus persica (L.) Batsch orPrunus davidiana (Carr.) Franch. (Fam. Rosaceae). The fruit is collectedwhen ripe. The seed is removed from sarcocarp and shell (endocarp), anddried in the sun.

Seed of Prunus Persica Prolate-ovate, 1.2-1.8 cm long, 0.8 1.2 cm wide,0.2-0.4 cm thick. Externally yellowish-brown to reddish brown, withnumerous granular protrudings. One end acute, expanded in the middle,the other end obtuse-rounded and slightly oblique with relatively thinedge. A short linear hilum occurring by the acute end and a relativelydistinct and slightly dark chalaza at the round end, with manylongitudinal vascular bundles radiated from the chalaza. Testa thin,cotyledons 2, almost white and oily. Odor weak; taste, slightly bitter.The medicinal preparations should be a pure Semen Persicae in powderform, clean and without impurities. Its important active ingredient isamygdalin.

Provider: Xingsheng Medicinal Herbs Company in Bozhou, Anhui P.R.C.Attn: L I, Yulong Telephone: 011-86-558-5525-258 Mobile:011-86-1395-6716-718.

Quality Control of Semi-Products

Creamed extractive: creamed extractive is dark brown in color. Itsrelative density should be no less than 1.27 (at 75˜80° C.), content ofpaeoniflorin should be 1.8˜2.7%

Soft powder after the mixing of Poria cortex: a yellowish brown incoloring with a slightly bitter taste. The microbe limit: number ofbacteria can't exceed 5000 entries/g; fungus can't exceed 300 entries/g;living acarid, acarid egg or coliform should not be detected. Content ofpaeoniflorin: 1.4˜2.2%.

Final granules: yellowish brown with water content no more than 4.0%

Capsules: clean, not sticky or mutilated in shape. Difference ofcontent: no more than 8.0% (0.31 g/capsule). Dissolving time is within25 minutes. Water content no more than 4.0%. Content of paeoniflorin isat 1.3˜1.9% and paeonol 0.7˜1.1%.

Yield Rate of Semi-Products

Poria powder 92~98% Soft material powder 94~98% Extract 15~18% Granule96~98%

Preparation of Compound Chinese Traditional Medicine Result in asophisticated physical and chemical procedures that involves tons ofcomplex compounds. During the decocting process, there always resultingin new ingredients. Base on properties of different medicine materialsin TCM and procedures of CCP, we have summarized general characteristicof various ingredients in CCP and charted them in the table below.

Components Content (%) β-Pachyman 32-36 Amygdalin 1.3 Amylum 30-34 Aminoacid 0.5 Salicylic acid 0.1 Dextrin 10-12 SiO₂ 0.5 H₂O 4 Colophony,shell Protein 4-6 and fat Tannin, β-cytellin Phlegm and glycose 10-14Paeoniflorin* 1.3-1.9 Paeonol* 0.7-1.1 *primary active componentSubsidiary ComponentsSilicon Dioxide

Other names: Silicon Dioxide; White Carbon; Silica Gel; Precipitatedsilica; Colloidal Silicon Dioxide.

Molecular formula and weight: SiO₂.nH₂O; 60.80 (anhydrous)

Preparative method: sodium silicate reacts with hydrochloric acid,nitric acid or vitriol to obtain silica gel which is aquiferous afterdepositing, washing, and drying. In air that is evenly mixed withhydrogen gas, silicon chloride hydrolyzes in water after burning underhigh temperature, and a solid aerosol is formed. Then use circulatingseparator to create anhydrous silica gel.

Properties: Due to differences in preparation methods, the product'sphysical & chemical characteristics vary. It is in white powder form andinsipid. With its hygroscopic feature, it does not resolve in water,organic solvent, or acid (except hydrofluoric acid). It does resolve inhot sodium hydroxide.

Usage: the product comes in tablet and capsule forms. It is used as adilutent, a supplement, an anti-adherent agent in preparation of tabletsor capsules; suspendsoid of ointment & suppository; stabilizer ofemulsion; absorbent of liquid components in solid preparation.Precipitable silica gel can also be used as a clarifying agent. Colloidsilicon dioxide can be used as the carrier of adsorbent solid dispersoidof organic substance with hydroxyl.

Stability and storage conditions: this product is hygroscopic, thereforeit should be stored in a container and put in a dry environment.

Dextrin

Molecular formula & weight: (C₆H₁₀O₅)n.xH₂O (162.14). Molecular weightis represented by n; average value is 4500.

Properties: this product is colored white and it's in powder or granuleform with a special fragrance and a sweet taste. Its bulk density is0.80; real density is 0.918; melting point is 178 (dissolved); watercontent is 5%; area of specific surface is 0.14 m²/g (quantasorb). It iseasily dissolved in hot water, and transforms into a clear colloidalsolution. It is also dissolvable in cold water, ethanol solution, orpropanol solution.

Usage: this product can be used as an absorbent, a dilutent, or abonding agent.

Properties of Some Important Ingredients

Paeonol

Chemical Name: Ethanone, 1-(2-hydroxy-4-methoxyphenyl)-

Molecular formula: C₉H₁₀O₃; Molecular weight: 166.17

Source: Cortex of Paeonia moutan Sim, family Ranunculaceae, herb ofPycnostelma paniculatum (Bunge) K. Schum, family Asclepiadaceae, bark ofBrtula platyphylla Suk. Var japonica (Sieb.) Hara, herb of Primulaauricula L, root of Rithysa meridionalis L. B. Smith & Downs.

Physical properties: colorless acerate crystal (ethanol), melting pointis 50° C., resolve a little in water, may be volatilized along withvapor, resolve ethanol, ether, acetme, chloroform, benzene and carbondisulfide.

Uvλ_(max) ^(EtOH) nm (log ε): 291 (4.61), 274 (4.17), 316 (3.84)Irν_(max) ^(KBr) cm⁻¹: 2940 & 1639 (strong). NMR (CDCL₃: 2.55 (3H, S),3.8 (3H, S), 6.3˜6.6 (2H, m), 7.6 (1H, d, J=9), 10.6 (1H, s). MSm/e (%):166 (M+, 42), 151 (100), 108 (8), 95 (7), 43 (8).

(TLC 1: silica gel G; Developer: cyclohexane-ethylester acetate (3:1);Coloration: 3% ferric chloride ethanol solution; Rf: 0.85 TCL 2: silicagel G; Developer: cyclohexane-chloroform-ethanol absolute (7:3:1);Coloration: 3% ferric chloride ethanol solution; Rf: 0.60)

Results of Current Studies on Physiological Activities:

1) On Central Nerves system: injected in a mouse or taken orally, andhave abirritative, antalgic & hypnotic function. It can reduce thetemperature and defervesce to a normal and calorifacient mouse, also itcould withstand convulsion induced by electricity and medicines. 2)Antibacterial and antiphlogistic Function: controlling consistency togolden staphylococcus and streptococcus faecalis is 500 μg/ml;controlling consistency to coliform and bacillus subtilis is 200 μg/ml,controlling consistency to tinea germ between toes is 250 μg/ml. Iftaking orally, it can withstand inflammation that was induced by carragcenin, glucosan and acetic acid. 3) Reducing Blood Pressure:anaesthetize dog and mainline 80˜120 mg/kg, reduce pressure 41˜61%, cankeep 10˜12 mins. It shows the obvious function of reducing pressure formost of the treated dogs and mouse with high blood pressure. 4)Toxicity: weak toxicity, mouse LD₅₀ (mg/kg), mainline 196, celiacinjection 741, take orally 3430 & 4900. 5) Clinic use: inject 50˜100 mgin muscle or certain points each time. It shows good curative effect totreat rheumatism, stomachache, other pains, eczema and irritabilitydermatitis etc., and the validity rate is 83%. Its validity rate is 92%to 261 cases with rheumatoid arthritis, lumbago, stomachache, bellyache,postoperative & cancer pain.

Paeoniflorin

Chemical Name: β-D-Glucopyranoside,5b-[(benzoyloxy)]tetrahydro-5-dydroxy-2-methyl-1,5-methano-1H-3,4-dioxacyclobuta[cd] pentalen-1a (2H)-yl [1aR-(1aα,2β,3aα,5α,5aα,5bα)]-

Molecular formula: C₂₃H₂₈O₁₁; Molecular weight: 480.45

Source: root of Paeonia albiflora Pall. (P. Lactiflora Pall.), root ofP. Suffruticosa Andr. (P. moutan Sims), root of P. Delavayi Franch.

Physical Properties: hygroscopic, formless powder, [α]_(D) ¹⁶ −12.8°(C=4.6, methanol), colorless & acerate crystal, the melting point is at196° C. UVλ_(max) ^(EtOH) nm (ε): 230 (9560). IRν_(max) ^(nujol) cm⁻¹:3400 (br.), 1708, 1604, 1585, 1270.

Results of Current Studies on Physiological Activities:

1) Function to Cordis Vessel: dilate coronary artery, the effect willincrease the flow of coronary artery, withstand acute lack blood ofcardiac muscle, control coacervation of blood platelet, reduce the bloodpressure etc, it has been tested out for treatment on coronary heartdisease. 2) Ease pain and calmness: it has obvious antalgic effect to ainjected celiacly mouse, can prolong the mouse's sleep time induced by,and can antagonize the convulsion induced by. 3) Antiinflaming andanticankerous function: can control ankle swelling induced by intradx,can prevent irritability ulceration, and restrain exudation of gastricjuice. 4) Antifebrile function: can reduce the normal animal heat ofmouse, can allay the animal heat of mouse creating fevered artificially.5) Antispastic function: can restrain the movement of exosomaticintestines and endosomatic stomach of big rat & guinea pig, and urinesmooth muscle of mouse. Can antagonize urine constriction of mouseinduced by alpha-hypophamine. 6) Toxicity: low toxicity, LD₅₀ (mg/kg) ofmouse: 3530 (mainline), 9530 (celiac injection).

Cinnamic Acid

Chemical Name: 2-Propenoic acid, 3-phenyl-

Molecular formula & weight: C₉H₈O₂; 148.16

Botanic source: Bark of Cinnamomum cassia Presl, family Lauraceae,velamen of Lycium Chinese Mill, leaves of Foeniculum vulgare Mill,family Umbelliferae, colophony of Myroxylon pereir (Royle) Klotzscli,family Leguminosae.

Physical properties: (convert) melting point 132˜134° C., UVλ_(max)^(EtOH) nm: 268, MS m/e 148 (M⁺, basal peal). Melting point 135˜136° C.,Boiling point 300° C.; UVλ_(max) ^(EtOH) nm (ε): 273 (20893), 222(14125), 216 (17783); Irν_(max) ^(KBr) cm⁻¹: 2520, 1690, 1680, 1640,1590, 1500, 1460, 1430, 1350, 1320, 1290, 1240, 1210, 1180, 1070, 980,940, 870, 770, 710, 590, 540, 480, MS m/e (%): 147 (100), 748 (94), 103(63), 77 (50), 51 (50), 102 (44), 131 (31), 91 (25)^([2]).

Results of Current Studies on Physiological Activities:

Antibacterialm and antifungal function, use as antiseptic. Be used totreat tuberculosis clinically, can increase leukocyte. In recent years,the animal testing proves that the function can increase leukocyte.Inject 1.5 mg/kg natrium cinnamate hypodermically to every rabbit, andkeep 3 days, increase 200˜250% leukocytes; keep over 10 days, has noinfluence to weight & temperature of animal. Has cholagogue function todogs; has slightly lax function.

Amygdalin

Other Names: Mandelonitrile-β-gentiobioside, Amygdaloside, laetrile,VB-17.

Chemical Names Benzeneacetonitrile,α-[(6-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]-,(R)—

Molecular Formula and Weight: C₂₀H₂₇NO₁₁; 457.42

Source: the seed of Prunus persica (.) Batsch. seed of P. amygdalusBatsch. seed of P. armeniaca L. seed of P. salicina Lindl. Kernel of P.mume (Sieb.) Sieb. et Zucc. branch of taegus oxyacantha Linn and Sorbustianschanica Rupr. seed of Eriobotrya japonica (Thunb.) Lindl, fruit ofCydonia oblonga Mill.

Physical Properties: The trihydrate is rhombic crystal (water), 200° C.melting point; melting point of anhydrous thing is about 220° C.,[α]_(D) ¹⁶ −42° C. 1 g resolves in 12 ml water, 900 ml ethanol and 11 mlboiling ethanol. It is easy to resolve in boiling water, hardly dissolvein ether. UVλ_(max) ^(Meon) nm (ε): 268 (179), 262 (255), 257 (256), 251(198), 208 (7210), IRν_(max) cm⁻¹, 3330, 2860, 1640, 1590, 1490, 1450,1270, 1160, 1060, 690^([1]).

Results of Current Studies on Physiological Activities:

It has been used for treatment on cancer since 1845. In recent years, ithas been often reported overseas. There are different parlances aboutits function and mechanism. Some think that it has anticancer functionsince β-glycuronidase of cancer tissue act on 1-β-glycuronic acid, andbring deadly HCN. Many Japanese and American reports shows that it hasobvious curative effect, but there are some contrary reports, and havecases which it resulted in death after taking. Therefore there isdispute to its anticancer value. At present time, America State CancerInstitute is treating clinically 200 cancer patients to which otherremedies take no effect.

Pachyman

During the past 10 years, more studies have been reported, since theresult showing obvious antitumor effects of Chtosan, Pachyman andAmorphalls mannan have brought on attention of researchers in the fieldsof organic chemistry, biochemistry, phytochemistry, physic and pharmacy.

Stability Test of CCP

Three batches of CCP (batch number: 960302, 960315, 960321) were testedof the stability at room temperature for 36 months. The following itemswere tested, among which the content of Paeonol was tested as an indexof the chief ingredients: appearance (product should be brownish yellowgranules, smells slightly herb aromatic and a little bitter in taste);identification of Cinnamomi and Poria (results showed as positive ornegative); the amount of Paeonol (%, method of HPLC); water contents orweight lost after dried in the thermostat (%); Hygienical inspection(number of bacterial, fungi and acarid). The result is show in table 1

TABLE 1 Stability test of CCP (room temperature) Time 0 months (1996.3)3 months (1996.6) Batch No. 960302 960315 960321 960302 960315 960321Appearance Brownish yellow granules, Brownish yellow granules, aromatic,slightly bitter aromatic, slightly bitter Cinnamomi + + + + + +Poria + + + + + + Disintegra- 7 7 9 7 7 7 tion(min) Weight Comply withspecified Comply with specified variation quality requirements qualityrequirements Water cont. % 5.1 5.4 4.8 5.1 5.4 4.8 Bacteria/g 20 30 2030 30 20 Fungi/g 10 20 10 20 20 20 Colibacillus Not tested Not testedAcarid Not tested Negative Paeonol (%) 0.78 0.876 0.80 0.78 0.76 0.80Time 6 months (1996.9) 12 months (1997.9) Batch No. 960302 960315 960321960302 960315 960321 Appearance Brownish yellow granules, Brownishyellow granules, aromatic, slightly bitter aromatic, slightly bitterCinnamomi + + + + + + Poria + + + + + + Disintegra- 8 8 9 9 7 8tion(min) Weight Comply with specified Comply with specified variationquality requirements quality requirements Water cont. % 5.2 5.4 4.9 5.15.4 4.8 Bacteria/g 30 30 20 30 30 20 Fungi/g 20 20 20 20 20 20Colibacillus Not tested Not tested Acarid Negative Not tested Paeonol(%) 0.78 0.76 0.80 0.77 0.76 0.81 Time 18 months (1997.9) 24 months(1998.3) Batch No. 960302 960315 960321 960302 960315 960321 AppearanceBrownish yellow granules, Brownish yellow granules, aromatic, slightlybitter aromatic, slightly bitter Cinnamomi + + + + + + Poria + + + + + +Disintegra- 9 8 8 8 8 9 tion(min) Weight Comply with specified Complywith specified variation quality requirements quality requirements Watercont. % 5.1 5.4 4.8 5.2 5.5 4.9 Bacteria/g 30 30 20 30 30 20 Fungi/g 2020 20 20 20 20 Colibacillus Not tested Not tested Acarid NegativeNegative Paeonol (%) 0.78 0.75 0.79 0.77 0.76 0.80 Time 30 months(1998.9) 36 months (1999.3) Batch No. 960302 960315 960321 960302 960315960321 Appearance Brownish yellow granules, Brownish yellow granules,aromatic, slightly bitter aromatic, slightly bitterCinnamomi + + + + + + Poria + + + + + + Disintegra- 9 7 8 9 8 8tion(min) Weight Comply with specified Comply with specified variationquality requirements quality requirements Water cont. % 5.2 5.5 4.9 5.25.5 4.9 Bacteria/g 30 30 30 30 30 20 Fungi/g 20 20 20 20 20 20Colibacillus Not tested Not tested Acarid Not tested Negative Paeonol(%) 0.77 0.75 0.80 0.77 0.76 0.79Fingerprinting of Capsule of Cinnamomi & Poria1) Gas Chromatography of CCP's Volatile Matter

System Requirement: HP-5 5% phenyl methyl siloxane capillary columns(30.0 m×0.32 mm×0.25 um); columns temperature 80° C. (5 min) to 250° C.(10 min) rising at 3° C./min; carrier gas is nitrogen with flow rate 1.5ml/min; FDI detector with hydrogen ml/min, air 350 ml/min; makeup gas:nitrogen 30 ml/min; inlet system temperature 250° C.; split injectionwith 50:1 in split ratio and 2 μl. in volume; temperature of detector280° C.; recording time 72 minutes.

Preparation of sample solution of volatile matter: Add 50 ml water and30 ml ethyl ether to the content of 10 capsules of Cinnamomi and Poria.Extract the solution with reflex on a Water bath of 75° C. for 90minutes and let it cool down. Extract the water layer of the solutionwith 20 ml ethyl ether for three times and merge all ethyl ethersolutions together. Volatilize to dry up the ethyl ether solution on awater bath of 35° C. and, finally, add ethyl ether to make it 5 ml involume.

Preparation of standard solution of cinnamaldehyde: Prepare ethyl ethersolution of cinnamaldehyde that contains 0.5 mg/ml of cinnamaldehyde.

System adjustment assay: 1) Number of theoretical plates: under designedcircumstance, inject 2 ul of Standard cinnamaldehyde solution into theGas Chromatography. Base on calculation on cinnamaldehyde peak, thenumber of theoretical plates should be no less than 800,000. 2) Systemaccuracy: Relative Standard Deviation (RSD) of the peak areas in fiveconsecutive injections of 2 ul standard cinnamaldehyde solution shouldbe no more than 3.0%.

TABLE 2 Peaks of volatile matter in CCP, Gas Chromatography; Standardpeak: cinnamaldehyde (self-comparison), peak 4: paeonol (see FIG. 1 forGC of volatile matter) Retention Range of Peak Number Time/AreaRetention Time Range of Aria 1 0.757/1.183 0.770~0.745 1.740~0.690Standard 1/1 2 1.275 1.280~1.270 3 1.290/0.696 1.295~1.285 1.110~0.350 41.449/5.565 1.455~1.440 8.080~3.460 5 1.704/0.093 1.710~1.7000.140~0.060 6 2.146/0.164 2.150~2.140 0.255~0.080 7 3.061/0.1153.070~3.055 0.160~0.0702) HPLC of CCP's Water-Soluble Matter

System Requirements: Spectrum column: Alltima C₁₈ 5 um, 7.5 mm×4.6 mm(guard column) and 250 nm×4.6 mm (spectrum column); column temperature30° C.; flow rate 1 ml/min, detective wavelength 230 nm; injectionvolume 5 ul; recording time 70 minutes.

Flow Phase: CH₃CN—H₂O—H₃PO₄

-   -   A 50:950:1    -   B 400:600:1    -   A        B

Preparation of sample solution of water-soluble matter: Add 200 ml waterto the content of 3 capsules. Extract the solution with reflex for 30minutes (after boiling) and let it cool down. Centrifuge the solutionfor 10 minutes and filtrate its supernatant with 0.45 um filter membraneto obtain the filtrate.

Preparation of standard paeoniflorin solution: Prepare methanol solutionof paeoniflorin that contains 0.5 mg/ml of paeoniflorin.

System adjustment assay: 1) Number of theoretical plates: under designedcircumstance, inject 2 ul of Standard paeoniflorin solution into theHPLC. Base on calculation on paeoniflorin peak, the number oftheoretical plates should be no less than 250,000. 2) System accuracy:Relative Standard Deviation (RSD) of the peak areas in five consecutiveinjections of 1 ul standard paeoniflorin solution should be no more than3.0%.

TABLE 3 Peaks of water-soluble matter, HPLC; Standard peak: paeoniflorin(self-comparison), peak 1: gallic acid, peak 4: albiflorin, peak 10:1,2,3,4,6-penta-O-galloyl-β-D-glucose, peak 13: mostly paeonol (see FIG.2 for HPLC of water-soluble matter) Retention Range of Peak NumberTime/Area Retention Time Range of Aria 1 0.261/0.645 0.275~0.2500.750~0.460 2 0.349/0.103 0.360~0.340 0.160~0.070 3 0.584/0.1280.600~0.560 0.230~0.065 4 0.915/0.212 0.920~0.910 0.250~0.170 Standard1/1 5 1.076/0.089 1.085~1.070 0.130~0.065 6 1.118/0.046 1.125~1.1100.060~0.035 7 1.162/0.052 1.175~1.155 0.080~0.030 8 1.196/0.0831.210~1.180 0.105~0.055 9 1.268/0.076 1.285~1.250 0.090~0.065 101.312/0.211 1.330~1.295 0.255~0.140 11 1.420/0.404 1.450~1.4000.470~0.310 12 2.107/0.149 2.170~2.060 0.195~0.130 13 2.389/0.9812.465~2.340 1.475~0.6803) HPLC of CCP's Liposoluble Matter

System Requirements: Spectrum column: Alltima C₁₈ 5 um, 7.5 mm×4.6 mm(guard column) and 250 nm×4.6 mm (spectrum column); column temperature50° C.; flow rate 1 ml/min, detective wavelength 210 nm and 242 nm;injection volume 5 ul; recording time 76 minutes.

Flow phase: CH₃CN—H₂O—H₃PO₄

-   -   A 600:400:1    -   B 950:50:1    -   A (25 min)        B (11 min)

Preparation of sample solution of liposoluble matter: Use theprecipitation deposit that obtained from after the centrifugal processin water-soluble solution preparation. Flush the deposit with wateruntil it becomes colorless. Add 20 ml methanol to the deposit, extractthe solution with reflex for 30 minutes (after boiling) and let it cooldown. Centrifuge the solution for 10 minutes. Volatilize to dry up themethanol from the solution on a water bath of 75° C. And finally, addcertain amount of methanol to make the solution to be at 2 ml in volume,filtrate it with 0.45 um filter membrane and obtain the filtrate.

Preparation of standard Poria raw material solution: Add 10 ml ofmethanol into 1 g. of Poria powder. Extract the solution with reflex for30 minutes (after boiling) and let it cool down. After centrifugal,filtrate the supernatant with 0.45 um filter membrane and obtain thefiltrate.

Standard Poria raw material is used in locating and identifying the twochemicals in liposoluble sample solutions: pachymic acid and polyporenicacid C at 210 and 242 nm respectfully. Pachymic acid and polyporenicacid C are chemicals within the samples and serve as standard peaks.System adjustment assay: Same with HPLC of Water-soluble matter

TABLE 4 Peaks of liposoluble matter, HPLC 210 nm; Standard peak:pachymic acid (self-comparison), peak 1 contains dehydrorumulosic acid,peak 6 contains 3 β-hydroxylanosta-7,9 (11), 24-trien-21-oic acid (seeFIG. 3 for HPLC, 210 mn of liposoluble matter) Retention Range of PeakNumber Time/Area Retention Time Range of Aria 1 0.367/0.322 0.375~0.3550.540~0.180 2 0.408/0.580 0.420~0.395 0.900~0.410 3 0.897/0.2800.905~0.890 0.350~0.220 4 0.980/0.752 0.985~0.975 0.940~0.600 Standard1/1 5 1.019/0.286 1.025~1.015 0.410~0.210 6 1.143/4.650 1.150~1.1355.950~2.900 7 1.305/0.959 1.315~1.295 1.450~0.575

TABLE 5 Peaks of liposoluble matter, HPLC 242 nm; Standard peak:polyporenic acid C (self-comparison), peak 3 contains dehydrotumulosicacid, peak 4 contains 3-epi-dehydrotumulosic acid, peak 6:dehydropachymic acid, peak 7 contains 3 β-hydroxylanosta-7,9 (11),24-trien-21-oic acid (see FIG. 4 for HPLC, 242 mn of liposoluble matter)Retention Range of Peak Number Time/Area Retention Time Range of Aria 10.520/0.155 0.530~0.510 0.185~0.120 2 0.566/0.184 0.570~0.5600.230~0.140 3 0.686/1.385 0.690~0.680 1.950~0.900 Standard 1/1 41.128/0.481 1.135~1.125 0.530~0.430 5 1.557/0.181 1.585~1.5250.240~0.150 6 1.763/1.414 1.800~1.720 1.700~1.250 7 2.133/0.6302.190~2.070 0.840~0.480 8 3.033/0.463 3.105~2.950 0.680~0.160Leading Pharmacodynamics Experiment about Remedial Effect of Capsule ofCinnamomi & Poria (CCP)

In order to determine the curative effects of the CCP, it is necessaryto apply experimental research on animals. The research with animalsshowed that the CCP relaxes the smooth muscles, help reduce the bloodviscosity, inhibit the agglutination of the blood platelet and it alsohas anti-inflammatory functions. Test on rats' extra-corporeal uterinecontraction, and the analgesic effects on live rats etc, were doneaccording to the leading pharmacodynamics research requirements fordiseases of hypercoaguability established in the “New ChineseTraditional Medicine Research Directory”, issued by the DrugAdministration of Chinese Sanitation Bureau.

Capsule of Cinnamomi & Poria causes an inhibitory effect on thecontraction of the smooth muscle of rats' extra-corporeal uterine. Usinga 2.7 mg/ml extract of CCP, it is possible to gain 100% control of thecontraction of the extra-corporeal uterine. Comparing the results to thecontrol group with normal saline, a smaller amount of CCP (ointmentformed extract 1.9 mg/ml, 1.3 mg/ml) can partly inhibit the smoothmuscle contraction frequency (P<0.05), range (P<0.05) and its overallactivity (p<0.001, p<0.01). The results showed there is a closedose-effect relationship between the concentration of CCP and theinhibitory effect.

Results also showed that a 5.4-mg/ml of CCP's extract might counteractthe effect of alphahypophamine, which at the same time may increase thefrequency of contraction of the extra-corporeal uterine of rats. Miceare administered the extract of CCP for 3 consecutive days. One hourafter the capsule has been administered on the third day, inject aceticacid in abdomen. Observe the twisting times of mice, which is a reactionto pain. The results showed that 0.63 g/kg or 1.26-g/kg of CCP's extractcan reduce the twisting times of mice, induced by showing a significantdifference (p<0.05, p<0.01) when compared with the negative controlgroup (normal saline) instead of CCP.

A high-dose of CCP (1.26 g/kg) resulted in a group's controlling rate oftwisting times of mice similar to that obtained from the positivecontrol group (aspirin group, 56.9% and 61.08% respectively). The highdose effect can also prolong the time mice swing their tales, which isalso a reaction to pain. Such experiments of analgesia demonstrated thatlike aspirin, the CCP had a definite analgesic effect.

Mice are administered the drug for 5 consecutive days. Half an hourafter the administration of the capsule on the fifth day, lightlyanaesthetize the mice with ether, sample rats' blood from the abdominalaorta to determine the Whole Blood Ratio Viscosity. The results showedthat by administering a 5.0 g/kg CCP, there is a decrease in the WholeBlood Ratio Viscosity. This explains the existence of a significantdifference when these results are compared to those obtained from thenormal saline control group (low shear P<0.01, high shear P<0.05). TheWhole Blood Ratio Viscosity continued to decrease when the dose wasincreased. The results from the experiment demonstrated theeffectiveness of the CCP when used to reduce the Whole Blood RatioViscosity of rats.

The results provided proof that a 12.5 mg/ml of CCP could reduce theconglomeration rate of blood platelet. There is a significant differencebetween these results and the ones obtained from the normal salinecontrol group (p<0.05). The conglomeration rate of blood plateletdecreased, as the dose was increased (CCP powder were 37.5 mg/ml and 75mg/ml respectively).

The experiments of conglomeration of blood platelet indicated that a 10g/kg extract of CCP reduced the conglomeration rate of blood platelet.The more the dose of CCP was increased, the stronger the effect. Theresults from the experiments about conglomeration of blood platelet alsoshowed that the CCP could inhibit the increase rate of conglomeration ofblood platelet induced by ADP.

Mice are administered the drug for three consecutive days. On the thirdday, smear an inflammatory inducing agent on the mice's right ear. Theanimals were killed two days after the smearing. The results indicatedthat the extract of CCP reduced the ear's swelling (P<0.01). Based onthe results obtained in this experiment, it was proved that the CCP hasa positive anti-inflammation effect. The research demonstrated that theCCP has acesodyne, antispastic and anti-inflammation effects. It coulddecrease the Whole Blood Viscosity, and inhibit the conglomeration ofblood platelet.

Purpose of the Experiment

The CCP has the following effects: releases the smooth musclecontraction of rats' extra-corporeal uterine, acts against the enhancedsmooth muscle contraction induced by oxytocin, reduces the twistingtimes of mice that was induced by acetic acid and prolongs mice'sswinging tail time caused by temperature. The CCP reduces the WholeBlood Ratio Viscosity, and inhibits the conglomeration rate ofextracorporeal blood platelet of rabbits. The CCP has definiteacesodyne, antispastic and anti-inflammatory effects. It also has theproperty to reduce Whole Blood Viscosity ratio, controls theconglomeration of blood platelet, and could be used for the treatment ofdysmenorrhea in clinical practice.

Uterine Contraction

The CCP can activate blood circulation and eliminate stasis. It ismostly used to treat symptoms of dysmenorrhea and disorders fromirregular shedding of endometrim, or many common pelvic disorders causedby smooth muscle movement or spasms in clinical practice in China. Thisresearch was done in order to establish the effects that the CCP has onuterine contraction and its possible curative effects.

Materials

Animal:

Female Wister rats with a weight of 180 g to 190 g. The rats weresupplied by The Experimental Animals Centre of Nanjing Railway MedicalInstitute (Certificate No. 97002, 97003). The rats had access to food,water and sunlight one whole week prior to the experiment.

Medicines:

Extract of CCP (1 g of CCP product corresponds to 4 g of the extract),Provided by Lianyungang Kanion Pharmaceutical Co. Ltd. (batch No.:990515).

Dihydrotheelin (2 mg/ml), Batch No: 971103. Produced by Shanghai 9^(th)Pharmaceutical Factory.

Oxytocin injection (10 u/ml), Batch No: 981228. Produced by Shanghaibiochemistry Pharmaceutical Factory.

Pregnendione. Provided by Dr Swerdoiff of LAC-UCLA Medical Center.

Equipment:

JZ100 Muscle Tension Transducer produced by Nanbeidian City XinhangElectro-mechanical Equipment Co. Ltd.

Yinhe 501 Type Superthermostat produced by Chongqing City ExperimentalEquipment Factory.

MS302 Physiological Information Processor provided by Pharmacology StaffRoom of Guangzhou Pharmacy Institute.

The data collected for this experiment was stored on a Pentium II 300.

Methods

Preparation of Drug:

a) Put 5.4 g extract of CCP in 100 ml of the Locke's solution and mixedthoroughly. The drug concentration results in 54 mg/ml. Take 1 ml ofthis solution, put it into a homothermic flume with 9 ml of the Locke'ssolution. The drug concentration from this mixture contains 5.4 mg/ml.Likewise, put 2.7 g extract of CCP in 100 ml of the Locke's solution andmixed thoroughly. Take 1 ml of this mixture, combine it with 9 ml ofLocke's solution and the drug concentration will result in 2.7 mg/ml.Mix up 100 ml of the Locke's solution with a 1.9 g CCP's extract. Take 1ml of this mixture, combine it with 9 ml of the Locke's solution and thedrug concentration will result in 1.9 mg/ml. Mix 200 ml of the Locke'ssolution with 2.6 g of CCP's extract, the concentration will result in13 mg/ml. Take 1 ml of this mixture, combine it with 9 ml of the Locke'ssolution which results in a drug concentration of 1.3 mg/ml. Mix 200 mlor 300 ml of the Locke's solution with 1.8 g CCP's resulting in a drugconcentration of 9 mg/ml and 6 mg/ml respectively. Take 1 ml of thismixture into homothermic flume with 9 ml of the Locke's solution. Theresult is a drug concentration containing 0.9 mg/ml and 0.6 mg/mlrespectively

Preparation of Locke's Solution

9.0 g of NaCL, 0.42 g of KCL, 0.24 g of CaCL₂, 0.2 g of NaHCO₃ and 1.0 gof glucose. Mixed with distilled water until obtaining 1000 ml. PH isabout 7.

Experiment Method

Twenty-four hours prior to the experiment, intramuscularly injectnon-pregnant female rats with a 0.3 mg/kg weight-dose of Dihydrotheelin.The effect artificially puts them in estrus period so as to increasetheir sensitivity to the CCP. Decapitate the rats, rapidly paunch, takeout the uterus and remove the fat. Cut the uterus in 2 cm of length andput it directly in the homothermic flume with 9 ml of the Locke'ssolution. One end of the uterus must be fixed on the crotchet of the Lshaped ventilating pipe and the other end connected to the MuscleTension Transducer. Keep the temperature of the homothermic flume at 32°C. Continuously emit oxygen into the fume, at least 60-80 bladders perminute and add in 1 ml of the previous solution with a differentconcentration. After recording a segment of normal contraction curve,observe the different phases before-and-after administering the drug.Respectively, record the contraction frequency and range. Observe from 1to 10, 10 to 20, and 20 to 30 minutes before-and-after adding the drugsolution. Count the uterine activity, and record data between controlgroups before-and-after administering the drug.

Observe CCP's effect in smooth muscle contraction of rats'extracorporeal uterine induced by oxytocin. Respectively, record thecontraction frequency and range. Observe from 1 to 10, 10 to 20, and 20to 30 minutes before-and-after adding the drug solution. Count theuterine activity and the inhibitory percentage. Record data aspreviously done.

Data Processing:

Activities = Frequency × Range${{Percentage}\mspace{14mu}{of}\mspace{14mu}{Inhibition}} = {\frac{\begin{matrix}{\begin{matrix}{{Frequency}\mspace{14mu}{before}\mspace{14mu}{administering}} \\\left( {{{or}\mspace{14mu}{range}},{activities}} \right)\end{matrix} -} \\\begin{matrix}{{Frequency}\mspace{14mu}{after}\mspace{14mu}{administering}} \\\left( {{{or}\mspace{14mu}{range}},{activities}} \right)\end{matrix}\end{matrix}}{\begin{matrix}{{Frequency}\mspace{14mu}{before}\mspace{14mu}{administering}} \\\left( {{{or}\mspace{14mu}{range}},{activities}} \right)\end{matrix}} \times 100\%}$

All data is shown with the average value ±standard deviation (X±SD); Alldata between groups and within every group is examined using the t valuein order to observe any significant difference.

Effect in Normal Rats' Uterine Smooth Muscle Contraction

Animals' Grouping: randomly divide the Wister female rats'extracorporeal uteruses into a normal saline control group (NSG) and aCCP group (CCPG). The dose of every group is respectively 2.7, 1.9, 1.3,0.9 and 0.6 mg/ml of CCP extract.

Results: under stable conditions, extra-corporeal uteruses can keepactive steadily within a period of 60 minutes. Under the 7 μg/mlconcentration, the pregnendione can control contraction frequency ofrats' uterine smooth muscle. Larger doses of extract of CCP (2.7, 1.9,1.3 mg/ml) can control the contraction frequency (See tables 2 and 3),the range (See tables 4 and 5) and the activity (See tables 6 and 7) ofrats' uterine smooth muscle in different degrees. A 2.7 mg/ml dose ofCCP's extract can stop contraction of the uterine smooth muscle. Theeffect the drug produces in the uterine smooth muscle contraction can benoticed ten minutes after the drug has been administered. The controlpercentage can get to 100% (p<0.001).

TABLE 6 Influence of CCP's extract to contraction frequency in rats'extra-corporeal uterine smooth muscle (time/10 mins) (X ± SD) BeforeAfter adm. After adm. After adm. Group Adm. 1-10 mins 0-20 mins 20-30mins NSG  7.5 ± 1.0 8.1 ± 0.7 8.4 ± 1.7 7.8 ± 0.9 Progestin  7 μg/ml 8.0 ± 0.8 6.1 ± 2.0 6.3 ± 2.5 5.0 ± 2.4* CCP 2.7 mg/ml  7.4 ± 1.4 1.7 ±1.5** 0*** 0*** 1.9 mg/ml  7.9 ± 2.7 5.0 ± 1.8 2.9 ± 3.9* 2.7 ± 3.6* 1.3mg/ml  8.2 ± 1.3 5.4 ± 1.4* 4.2 ± 2.4* 4.1 ± 3.0 0.9 mg/ml 10.8 ± 2.97.8 ± 1.7 7.8 ± 2.4 7.6 ± 4.4 0.6 mg/ml  8.3 ± 3.1 6.9 ± 2.3 8.3 ± 3.38.0 ± 3.9 *p < 0.05 **p < 0.01 ***P < 0.001 (compare with itself beforeadministering)

TABLE 7 Influence of CCP's extract on the contraction frequency in rats'extra-corporeal uterine smooth muscle (Controlling percenage %) Afteradm. After adm. After adm. Group 1-10 mins 10-20 mins 20-30 mins NSG −8−12.0 −5.3 Progestin  7 μg/ml 23.8 21.3 37.5 CCP 2.7 mg/ml 77.0 100.0100.0 1.9 mg/ml 36.7 63.3 65.8 1.3 mg/ml 34.1 48.9 50.0 0.9 mg/ml 27.827.8 29.6 0.6 mg/ml 16.9 0 3.6

TABLE 8 Influence of CCP's extract on the contraction range of rats'extra-corporeal uterine smooth muscle (mm) Before After adm. After adm.After adm. Group Adm. 1-10 mins 10-20 mins 20-30 mins NSG  8.1 ± 2.1 8.3± 1.7 8.4 ± 2.0 8.4 ± 2.5 Progestin  7 μg/ml  8.6 ± 2.2 7.4 ± 2.3 7.0 ±2.6 7.0 ± 2.8 CCP 2.7 mg/ml 10.8 ± 1.3 7.4 ± 5.2 0.0 ± 0.0*** 0.0 ±0.0*** 1.9 mg/ml  9.5 ± 1.8 7.0 ± 2.2 2.8 ± 2.7** 1.7 ± 2.4** 1.3 mg/ml10.4 ± 1.9 7.5 ± 2.0* 5.3 ± 2.2* 4.9 ± 2.4* 0.9 mg/ml  9.2 ± 1.9 8.7 ±1.9 8.1 ± 1.5 7.8 ± 1.4 0.6 mg/ml  8.8 ± 2.3 8.8 ± 2.3 8.7 ± 2.7 9.3 ±2.5 *p < 0.05 **p < 0.01 ***P < 0.001 (Compare with itself beforeadministering)

TABLE 9 Influence of CCP's extraction on the contraction range of rats'extracorporeal uterine smooth muscle (Controlling percent %) After adm.After adm. After adm. Group 1-10 mins 10-20 mins 20-30 mins NSG −2.5−3.7 −6.2 Progestin  7 μg/ml 14.0 18.6 18.6 CCP 2.7 mg/ml 31.5 100 1001.9 mg/ml 26.3 70.5 82.1 1.3 mg/ml 27.9 49.0 52.9 0.9 mg/ml 5.4 12.015.2 0.6 mg/ml 0 1.1 -5.7

TABLE 10 Influence of CCP's extract on the contraction activity of rats'extra-corporeal uterine smooth muscle (%) (X ± SD) Before After adm.After adm. After adm. Group adm. 1-10 mins 10-20 mins 20-30 mins NSG61.5 ± 22.9 67.9 ± 17.7 72.2 ± 26.2 65.7 ± 18.3 Progestin  7 μg/ml 69.3± 20.9 47.2 ± 22.9 46.3 ± 27.9 35.2 ± 22.5 CCP 2.7 mg/ml 79.9 ± 15.916.7 ± 13.2 0*** 0*** 1.9 mg/ml 74.1 ± 29.8 34.6 ± 14.0* 13.2 ± 16.6** 7.9 ± 10.2** 1.3 mg/ml 85.6 ± 19.0 41.5 ± 18.4* 23.6 ± 18.1* 23.2 ±27.5* 0.9 mg/ml 99.0 ± 36.2* 65.4 ± 12.0 63.5 ± 26.4 58.6 ± 33.2 0.6mg/ml 71.7 ± 31.8 59.9 ± 24.8 70.0 ± 33.1 73.6 ± 40.9 *p < 0.05 **p <0.01 ***P < 0.001

TABLE 11 Influence of CCP's extract on the contraction activity of rats'extra-corporeal uterine smooth muscle (Controlling %) After adm. Afteradm. After adm. Group 1-10 mins 10-20 mins 20-30 mins NSG −10.4 −17.4−6.8 Progestin  7 μg/ml 31.9 33.2 49.2 CCP 2.7 mg/ml 79.1 100 100 1.9mg/ml 53.3 82.2 89.3 1.3 mg/ml 51.5 72.4 72.9 0.9 mg/ml 33.9 35.9 40.10.6 mg/ml 16.5 2.4 -2.6

The results showed that the extract of CCP could inhibit the contractionof normal rats' uterus. A dose of 0.6-2.7 mg/ml of CCP determines theimproving effects of the capsule with the increasing of the dose. Thisaccounts as a dose-effect relationship.

CCP's Effect on the Contraction of Uterine Smooth Muscle Induced byOxytocin

Animals' Grouping: randomly divide Wister female rats' extra-corporealuteruses into normal saline control group (NSG) and CCP's group. TheCCP's group is formed by 4 subgroups with seven uteruses each. The doseof CCP's extract is respectively 5.4, 2.7, 1.3, and 0.6 mg/ml.

Results: after adding 10 mu/ml oxytocin to the normal uterine smoothmuscle there is an increase of the contraction frequency and the uterineactivity. The contraction range is also affected reaching its peak forabout 10 min to then stabilize on the level. After 40 minutes, theeffect gradually drops. After the oxytocin reacts for 10 minutes, add ina different dose of CCP's extract, and compare the possible differencesbetween the CCP's group and the oxytocin's group. A 5.4, 2.7 mg/ml doseof CCP's extract can control the increase of uterine activity induced byoxytocin, and mainly reduce contraction frequency of uterine smoothmuscle (See Table 8 and 13).

TABLE 12 Influence of CCP's extract on the contraction frequency ofuterine smooth muscle induced by oxytocin Before After adm. After adm.After adm. Group adm. 1-10 mins 10-20 mins 20-30 mins NSG 7.5 ± 1.0  8.4± 1.7  7.8 ± 0.9  7.3 ± 1.5 Oxytocin 7.0 ± 1.3 11.7 ± 1.5 11.6 ± 1.910.9 ± 1.17 Oxytocin + CCP 5.4 mg/ml 8.5 ± 1.6  6.6 ± 0.9*  5.3 ± 1.0** 3.4 ± 1.8** 2.7 mg/ml 7.9 ± 1.6  8.9 ± 1.3*  8.8 ± 1.3*  7.8 ± 1.0* 1.3mg/ml 8.4 ± 2.8 11.1 ± 1.6 11.6 ± 3.1 10.6 ± 2.6 0.6 mg/ml 9.2 ± 1.5*13.3 ± 2.0 12.4 ± 3.2 11.5 ± 2.3 *p < 0.05 **p < 0.01 ***P < 0.001(compare with relevant phase's frequency of oxytocin group)

TABLE 13 Influence of CCP's extraction to the contraction frequency ofuterine smooth muscle induced by oxytocin (Controlling %) After adm.After adm. After adm. Group Oxytocin 1-10 mins 10-20 mins 20-30 mins NSG−8 −12 −5.3 2.7 Oxytocin −91.4 −67.1 −65.7 −55.7 Oxytocin + CCP 5.4mg/ml −83.5 20.0 37.6 60.0 2.7 mg/ml −110.1 −12.7 −11.4 1.3 1.3 mg/ml−113.1 −32.1 −38.1 −26.2 0.6 mg/ml −77.2 −44.6 −34.8 −25.0

TABLE 14 Influence of CCP's extract on the contraction range of uterinesmooth muscle induced by oxytocin (mm) (X ± SD) Before After adm. Afteradm. After adm. Group adm. Oxytocin 1-10 mins 10-20 mins 20-30 mins NSG8.1 ± 2.1 8.3 ± 1.7  8.4 ± 2.0  8.6 ± 2.5  7.6 ± 1.5 Oxytocin 8.0 ± 2.67.3 ± 3.2  9.7 ± 5.0 10.4 ± 5.7  9.7 ± 6.1 Oxytocin + CCP 5.4 mg/ml 7.4± 0.9 7.2 ± 2.2  7.3 ± 1.1  6.7 ± 1.2  5.8 ± 1.7 2.7 mg/ml 8.9 ± 2.7 7.5± 2.8  9.5 ± 2.7 10.3 ± 3.3  9.7 ± 2.7 1.3 mg/ml 9.2 ± 2.3 8.6 ± 2.310.2 ± 1.8 10.6 ± 1.8 10.9 ± 2.3 0.6 mg/ml 8.5 ± 2.2 7.1 ± 3.7  8.7 ±5.2  8.9 ± 5.1  9.1 ± 5.7 *P < 0.05 **P < 0.01 ***P < 0.001 (comparewith oxytocin Group)

TABLE 15 Influence of CCP's extract on the contraction range of uterinesmooth muscle induced by oxytocin (controlling %) Before After adm.After adm. After adm. Group adm. 1-10 mins 10-20 mins 20-30 mins NSG−2.5 −3.7 −6.2 6.2 Oxytocin 8.8 −21.3 −30.0 −21.3 Oxytocin + CCP 5.4mg/ml 4.1 1.4 9.5 21.6 2.7 mg/ml 15.7 −6.7 −15.7 −9.0 1.3 mg/ml 6.5−10.9 −15.2 −18.5 0.6 mg/ml 16.5 −2.4 −4.7 −7.1

TABLE 16 Influence of CCP's extract on the contraction activity ofuterine smooth muscle induced by oxytocin (%) (X ± SD) Before After adm.After adm. After adm. Group adm. Oxytocin 1-10 mins 10-20 mins 20-30mins NSG 61.5 ± 22.9  67.9 ± 17.7  72.2 ± 26.2  65.7 ± 18.3  55.4 ± 17.9Oxytocin 56.9 ± 21.6  84.7 ± 22.0 118.8 ± 77.6 129.6 ± 97.3 111.2 ± 86.5Oxytocin + CCP 5.4 mg/ml 62.4 ± 11.3 111.7 ± 39.0  48.7 ± 12.6  6.2 ±12.8*  21.5 ± 16.2* 2.7 mg/ml 71.0 ± 27.5 124.3 ± 48.8  84.7 ± 23.7 90.6 ± 32.2  75.9 ± 22.7 1.3 mg/ml 79.3 ± 34.4 157.6 ± 61.1 112.3 ±18.4 119.9 ± 30.1 111.3 ± 12.6 0.6 mg/ml 79.1 ± 29.3 127.3 ± 92.8 122.9± 92.9 123.2 ± 115.0 113.7 ± 103.1 *p < 0.05 (compare with Oxytocingroup)

TABLE 17a Influence of CCP's extract on the contraction activity ofuterine smooth muscle induced by oxytocin (controlling %) After adm.After adm. After adm. Group Oxytocin 1-10 mins 10-20 mins 20-30 mins NSG−10.4 −17.4 −6.8 9.9 Oxytocin −48.9 −108.8 −127.8 −95.4 Oxytocin + CCP5.4 mg/ml −79.0 22.0 42.0 65.5 2.7 mg/ml −75.1 −19.3 −27.6 −6.9 1.3mg/ml −98.7 −41.6 −51.2 −40.1 0.6 mg/ml −60.9 −55.4 −55.8 −43.7

TABLE 17b Influence of CCP's extract on the contraction activity ofuterine smooth muscle induced by oxytocin Concentration (mg/ml) ControlRate (%) 3  9.06 ± 5.78 5.6 32.39 ± 9.15 10 53.63 ± 20.78 17.78 85.63 ±5.91 30 96.01 ± 2.8

The result showed that after administering 5.4 mg/ml of extract of CCPfor ten minutes, its effect produced a reduction of the contractionfrequency of the uterine smooth muscle.

Analgesia Test

Acetic Acid Twisting Experiment

The CCP is mostly used to treat dysmenorrhea, and to release the smoothmuscle of uterine. In order to prove the curative effects the drug hasin the treatment of dysmenorrhea, research has been done according tothe leading pharmacodynamics research requirements of the ChineseTraditional Medicine established in the “New Chinese TraditionalMedicine Research Directory”.

Materials

Animal: Use both female and male mice, 18 g to 23 g of weight. TheExperimental Animals Center of Nanjing Railway Medical Institute was incharge of providing the animals for this experiment. The rats haveaccess to food, water and sunlight one whole week prior to theexperiment.

Medicines: Extract of Capsule of Cinnamomi & Poria (1 g of CCP productcorresponds to 4 g of the extract). This extract is provided byLianyungang Kanion Pharmaceutical Co. Ltd (batch No.: 990515).

Acetylsalicylic acid, 25 mg/tablet, produced by Nanjing HengshengPharmaceutical Co. ltd.

Glacial acetic acid (CH₃COOH), concentration is 99.0%, produced byNanjing Chemical Reagent Factory.

Methods

Randomly divide the animals into 5 groups. Every group has twelve rats(7 female, 5 male). Groups A, B and C are administered a 0.18 g/kg, 0.63g/kg and 1.26 g/kg of extract of CCP respectively, once everyday forthree consecutive days. Group D is administered the normal saline at 0.2ml per mouse once everyday for three consecutive days. Group E isadministered 0.2 g/kg of glacial acetic acid (Aspirin) once everyday forthree consecutive days. The volume of the solution is 0.2 ml/time. Afterthe drug has been administered on the third day, celio-inject 0.5%acetic acid to the mice in groups. Observe and record the times oftwisting of mice within 30 minutes.

${{Controlling}\mspace{14mu}{Percent}} = {\frac{\begin{matrix}{{{Twisting}\mspace{14mu}{times}\mspace{14mu}{of}\mspace{14mu}{NSG}} -} \\{{Twisting}\mspace{14mu}{times}\mspace{14mu}{of}\mspace{14mu}{CCPG}}\end{matrix}}{{Twisting}\mspace{14mu}{times}\mspace{14mu}{of}\mspace{14mu}{NSG}} \times 100\%}$

All data is shown with a mean±standard deviation (X±SD). Examine thesignificance difference between groups using the t test.

Experimental Results

The effect of the Celiac-injection of acetic acid can result in atypical body reaction in response to the stimulus. Mostly, there appearsto be a twisting of the mice's bodies that includes an extension of hindlegs and hip rising, belly sinking and lumbar bending lateral. Both theaspirin groups and the CCP group reduced the twisting times to adifferent extent. An obvious difference arises if compare with thenormal saline control group. The results are shown in Table 14.

TABLE 18a Influence of the extract of CCP on the twisting responses ofmice induced by 0.5% acetic acid (times) Times of twisting ControllingGroup & dose Animal No. (0~30 mins) (%) NS 0.20 ml/mouse 12 38.8 ± 17.2CCP 0.18 g/kg 12 29.6 ± 14.7 23.7 CCP 0.63 g/kg 12 20.5 ± 15.0* 47.2 CCP1.26 g/kg 12 16.7 ± 13.6** 57.0 CCP 0.20 g/kg 12 15.1 ± 17.1** 61.1 *p <0.05, **p < 0.01(compare with NSG)

TABLE 18b Influence of the extract of CCP on the twisting responses ofmice induced by acetic acid (times) Times of Times of Times of Dosageno. of twisting twisting twisting Drag (g/Kg) Animal Adm. 0-10 10-200-20 Contral — 10 P.O 22.7 ± 4.4 23.6 ± 6.6 46.3 ± 7.3 CCP 2.5 10 P.O19.3 ± 2.5* 19.1 ± 6.6 38.4 ± 5.1* CCP 5.0 10 P.O 14.0 ± 6.0* 12.9 ±6.6** 26.9 ± 11.7 CCP 10.0 10 P.O 10.8 ± 6.6***  9.6 ± 7.3***  9.1 ±14.5 CCP 0.1 10 P.O  2.8 ± 1.4 *p < 0.05, **p < 0.01(compare with NSG)Test of Mice's Swinging TailMaterials

Animal: both female and male mice are grown Kunming, 18 g-23 g weight ofbody. The mice were supplied by the Experimental Animals Center ofNanjing Railway Medical Institute (Certificate No. Sudongzhizi-97002 &Sudonghuanzi-97003). The rats have access to food, water and sunlightone whole week prior to the experiment.

Medicines:

Extract of Capsule of Cinnamomi & Poria (1 g extract corresponds to 4 gof crude drug), The capsule is provided by Lianyungang KanionPharmaceutical Co. Ltd (batch No.: 990515).

Methods

Randomly divide the animals into 4 groups. Every group has 10 rats (fivefemale, five male). Groups A, B and C are administered a 0.18 g/kg, 0.63g/kg and 1.26 g/kg doses of extract of CCP respectively. Group D isadministered normal saline at 0.2 ml per mouse. The volume of thesolution is 0.2 ml/time. Immerge one third of the mice's tails in waterat 55° C. Record the swinging time of the mice's tales beforeadministering the drug. Then, administered the drug in different dosagesto the four groups and check the swinging time of mice's tale under theeffect of the drugs. Record the time of swinging for one, two and fourhours after the administration of the drug.

${{Controlling}\mspace{14mu}(\%)} = {\frac{\begin{matrix}{{{Swinging}\mspace{14mu}{tail}^{\prime}s\mspace{14mu}{latent}\mspace{14mu}{time}\mspace{14mu}{of}\mspace{14mu}{CCP}} -} \\{{Swinging}\mspace{14mu}{tail}^{\prime}s\mspace{14mu}{latent}\mspace{14mu}{time}\mspace{14mu}{of}\mspace{14mu}{NSG}}\end{matrix}}{{Swinging}\mspace{14mu}{tail}^{\prime}s\mspace{14mu}{latent}\mspace{14mu}{time}\mspace{14mu}{of}\mspace{14mu}{CCP}} \times 100\%}$

All data is shown with the mean±standard deviation (X±SD). Examine thedifferent significance between groups using the t test.

TABLE 19a Influence of CCP on mice's swinging tail latent time Afteradm. After adm. After adm. Group & dose Before adm. 60 mins 120 mins 240mins Normal saline 1.66 ± 0.46 1.56 ± 0.51 1.62 ± 0.46 1.65 ± 0.36 0.18g/kg 1.63 ± 0.42 1.73 ± 0.48 (5.8) 1.83 ± 0.49 (10.9) 1.83 ± 0.52 (10.9)0.63 g/kg 1.64 ± 0.34 2.18 ± 0.50*_(ΔΔ) (24.8) 1.97 ± 0.50_(Δ) (16.8) .10 ± 0.56_(ΔΔ) (21.9) 1.26 g/kg 1.58 ± 0.28 2.40 ± 0.61**_(ΔΔΔ) (34.2)2.34 ± 0.68**_(ΔΔΔ) (32.5) 2.54 ± 0.69**_(ΔΔΔ) (37.8) p < 0.05 **p <0.01(compare with before administering) _(Δ)p < 0.05 _(ΔΔ)p < 0.01_(ΔΔΔ)p < 0.001 (compare with control group)

TABLE 19b Influence of CCP on mice's swinging tail latent time DosesBefore After Adm. After Adm. After Adm. Drag (g/Kg) Adm. Adm. 60 mins120 mins 240 mins Ctrl. — 10 1.58 ± 0.43 1.49 ± 0.4 1.57 ± 0.38 1.54 ±0.41 CCP 5 10 1.56 ± 0.40 1.71 ± 0.37 1.91 ± 0.58 1.89 ± 0.66 CCP 10 101.55 ± 0.46 2.21 ± 0.93* 2.42 ± 0.95** 2.70 ± 0.92*** CCP 20 10 1.50 ±0.47 2.57 ± 0.95** 2.61 ± 1.27 2.50 ± 0.43*** XYT 0.01 10 1.58 ± 0.792.23 ± 0.53* 2.56 ± 0.40 1.52 ± 0.56 *P < 0.05, **P < 0.01, ***P < 0.001

The experiments' results showed that a 0.63 g/kg, 1.26-g/kg extract ofCCP could obviously prolong mice's time of tail swinging. Differenceswere noticeable when the results were compared to the normal salinecontrol group.

Conclusion

The experiment about the influence of the extract of CCP in mice'stwisting times showed that a 0.18 g/kg extract of CCP is effective inreducing the time range of the mice's twisting. The more the dose wasincreased (0.63 g/kg, 1.26 g/kg), the more the mice's twisting timesdecreased. Control rates are respectively 23.7%, 47.2% and 56.9% within1 to 30 minutes. There is a significant difference with the normalsaline control group. The control rate of the positive control-AspirinGroup is 61.08%. Since a 0.63 g/kg, 1.26 g/kg extract of CCP canobviously prolong the mice's twisting times. The results demonstratethat the CCP has strong analgesic effects on live rats.

Declining Whole Blood Viscosity

Materials

Animal: male Wister rats.

Medicines:

Extract of CCP (1 g extract corresponds to 4 g crude drugs) provided byLianyungang Kanion Pharmaceutical Co. Ltd. (batch No.: 990515).

Persantine, produced by Harbin White Swan Pharmaceutical Factory (batchNo.: 8905010).

Heparin, produced by Suzhou Xinbao Pharmaceutical Factory (batch No.:881105).

Equipment: XN-5 Type Blood Viscometer

Methods

Drug dispensation: put a 12.5 g extract of CCP in a 100 ml solution ofnormal saline and mix adequately. The drug concentration is 0.125 g/ml(containing 0.5 g/ml of crude drugs). Put a 25 g extract of CCP in 100ml of normal saline. The drug concentration is 0.25 g/ml (containing 0.1g/ml of crude drugs). Put a 50 g extract of CCP in a 100 ml of normalsaline and mix adequately. The drug concentration is 0.5 g/ml(containing 2 g/ml crude drugs).

Persantine: put 2 g in normal saline and mix adequately. The drugconcentration will be 20 mg/ml.

Experimental processing: Randomly divide male Wister rats weighing 250to 300 g into five groups with eight rats in each group. Low, medium andhigh dose groups can take the extract of CCP in a concentration of 10ml/kg. The concentration of crude drugs is 5 g/kg, 10.0 g/kg and 20 g/kgrespectively. The control group takes normal saline of the same volume;positive medicine group takes 0.2 g/kg, which is 10 ml/kg of persantine.The drug must be administered once a day for five consecutive days.After an hour and a half of administering the drug on the fifth day,lightly anaesthetize the mice with ether. Record 40 test tubes, and put0.1 ml heparin in each. Take blood from the abdominal aorta of the miceand fill each tube with 1 ml of blood. Then use XN-5 Type BloodViscometer to determine the Whole Blood Ratio Viscosity.

Data processing: All data is shown with the mean±standard deviation(X±SD), and the t test.

Results

The CCP's influence on the Whole Blood Viscosity of rats showed that a 5g/kg extract of CCP could reduce the Whole Blood Viscosity to minimumlow shear of P<0.01, and a maximum high shear of P<0.05. The Whole BloodViscosity continued to drop along with the increased of the dose. TheCCP can clearly reduce rats' Whole Blood Ratio Viscosity (see Table 16).

TABLE 20 Influence of CCP on the rats' Whole Blood Ratio Viscosity DoseAnimal Whole Blood Ratio Viscosity Medicine (g/kg) No. low shear highshear Normal saline 0 8 29.85 ± 9.33 12.10 ± 1.90 CCP 5.0 8 17.98 ±4.78**  9.66 ± 1.47* 10.0 8 16.69 ± 4.86**  8.49 ± 1.81** 20.0 8 15.36 ±4.38**  5.60 ± 2.15** Persantine 0.2 8 16.12 ± 6.92** 12.10 ± 1.90 Note:compare with the normal saline control group, *P < 0.05, **P < 0.01,***P < 0.001.Discussion

The Whole Blood Ratio Viscosity is an index that measures the viscosityof blood. It's always related to the quantity and quality of bloodcorpuscle, plasma cholesterol, and fibrin macromolecular concentration.Whole Blood Ratio Viscosity is the ratio of Whole Blood Viscosity andaqueous viscosity. The more blood corpuscles are found the higher theWhole Blood Viscosity becomes. The increase of the Whole Blood RatioViscosity changes the blood flow as a whole, including the change ofcorporeal soluble components in blood. This reaction is most affected byRBC. When RBC aggregates and agglomerates, the blood viscosityincreases, creating great resistance to the blood flow, a condition ofhigh viscosity blood named “hematogenous abnormality”. Because the CCPreduces blood viscosity, it is used to prevent and cure blood stasis.The Whole Blood Viscosity reflects the changes of the blood flow as awhole. Thus, the CCP can treat and improve symptoms of blood stasis.

Experiments about Extra-Corporeal Platelet Aggregation of Rabbit

Materials

Animal: Use live rabbits that weight 2.5 kg.

Medicine:

Extract of Cinnamomi & Poria (1 g extract corresponds to 4 g of crudedrugs). The extract is supplied by Lianyungang Kanion Pharmaceutical Co.Ltd. (No. 892011).

Bolus of Cinnamomi & Poria (1 g Bolus corresponds to 4 g of crudedrugs). The drug is supplied by Shanxi Changling Chinese TraditionalMedicine Factory (No. 900111).

Aspirin, produced by Shanghai Jiufu Pharmaceutical Company (No. 900111).

Instrument: SPA-Type Platelet Aggregation instrument.

Test Preparation

Preparation of drug: Put 3.13 g of extract of CCP in 100 ml of normalsaline. The drug concentration is 31.3 mg/ml (contains 125 mg/ml ofcrude drug). Take 1 ml from this solution, put it in 9 ml of normalsaline, and mix it adequately. The drug concentration is 3.13 mg/ml(contains 12.5 mg/ml of crude drug). Put 2 ml of the solution madepreviously, which contains the CCP, into 8 ml of normal saline, and mixit adequately. The drug concentration is 6.26 mg/ml (contains 25 mg/mlof crude drug). Put 3 ml of the solution made with the CCP in 7 ml ofnormal saline, and mix it adequately. The drug concentration is 9.39mg/ml (contains 37.5 mg/ml of crude drug). Put 4 ml of the solution madewith the CCP in 6 ml of normal saline, and mix it adequately. The drugconcentration is 12.5 mg/ml (contains 50 mg/ml of crude drug). Put 6 mlof the solution made with the CCP in 4 ml of normal saline, and mix itadequately. The drug concentration is 18.78 mg/ml (contains 75 mg/ml ofcrude drug).

Put a 1.5 g Bolus of Cinnamomi & Poria in 100 ml of normal saline, andmix it adequately. This solution contains 60 mg/ml of crude drug. Take 3ml from this solution, put in 7 ml of normal saline, and mix itadequately. The solution contains 18 mg/ml of crude drug. Then put 6 mlof the solution that contains the PCP in 4 ml of normal saline, and mixit adequately. This solution contains 36 mg/ml of crude drug. Put 9 mlof the solution that contains the PCP in 1 ml of normal saline. Thesolution contains 54 mg/ml of crude drug. Put 0.3 g of aspirin in 1000ml of normal saline, and mix it adequately. The solution contains 0.3mg/ml of crude drug.

Methods

a) Put 0.1 ml of 3.13% sodium citrate in 65 tubes.

b) Intubate and sample the blood of the rabbits taken from the commoncarotid artery while the animals are awake. Every tube should have 0.9ml of blood. The ratio between whole blood and anticoagulant is 9:1.Centrifugalize at 1000 rpm for seven minutes and prepare PRP. After thesuction of PRP, centrifugalize 10 min in terms of 3000 rpm to preparePPP. According to the Born method, use the SPA-III Type PPP PlateletAggregation instrument for the test. Different concentrations of CCP arefound: 12.5 mg/ml, 25 mg/ml, 37.5 mg/ml, 50 mg/ml and 75 mg/ml. Alsodifferent concentrations of PCP are found: 18 mg/ml, 30 mg/ml, 36 mg/ml,and 54 mg/ml. Prepare PRP using incubation for 10 min. Administer thenormal saline from the same volume to the control group. Administer theaspirin to positive control group. The concentration of ADP will be 1μm.

Data Processing

${{Control}\mspace{14mu}\%} = \frac{\begin{matrix}{{{Platelet}\mspace{14mu}{agregation}\mspace{14mu}{ratio}\mspace{14mu}{Before}\mspace{14mu}{administering}} -} \\{{Platelet}\mspace{14mu}{aggregation}\mspace{14mu}{ratio}\mspace{14mu}{after}\mspace{14mu}{administering}}\end{matrix}}{{Platelet}\mspace{14mu}{aggregation}\mspace{14mu}{ratio}\mspace{14mu}{before}\mspace{14mu}{administering}}$

All data is shown with the mean±standard deviation (X±SD). All databetween groups and within every group is examined using the t value inorder to observe any significant difference. Experimental results areshown in Table 17.

TABLE 21 Influence of CCP on the extra-corporeal platelet aggregation ofrabbits Concentration Sample Drugs (Mg/ml) No. Aggregation (%) Control(%) NS 0 5 67.95 ± 19.32 CCP 12.5 5 51.32 ± 11.78** 27.47 ± 7.76 25 539.60 ± 12.23** 41.72 ± 7.96 37.5 5 25.56 ± 9.95*** 62.30 ± 6.54 50 513.74 ± 5.32*** 79.78 ± 3.50 75 5  3.18 ± 2.21*** 95.33 ± 1.46 Aspirin0.3 5 32.94 ± 1.68** NS 0 5 66.17 ± 1.07 BP 18 5 57.83 ± 4.83* 32.60 ±7.305 30 5 34.50 ± 6.78**  7.86 ± 10.25 36 5 29.34 ± 7.34** 55.67 ±11.09 54 5  4.50 ± 0.79*** 93.20 ± 1.19 *P < 0.05, **P < 0.01, ***P <0.001(compare with NSG)Platelet Aggregation Experiments of RatsMaterials

Animal: male Wister rats that weight between 250 to 300 mg. The ratshave access to food, water and sunlight one whole week prior to theexperiment.

Medicine:

Extract of Capsule of Cinnamomi & Poria (1 g of extract corresponds to 4g of crude drug), are provided by Lianyungang Kanion Pharmaceutical Co.Ltd, Batch No. 891011.

Aspirin, supplied by the Shanghai Jiufu Pharmaceutical Co., Batch No.900111.

Instrument: SPA-III Type Platelet Aggregation instrument.

Method

Preparation of drug: Put 12.5 g of extract of CCP in 100 ml of normalsaline, and mix adequately. The drug concentration is 0.5 mg/ml. Put 25g extract in 100 ml of normal saline, and mix adequately. The drugconcentration is 1 g/ml. Put 50 g of extract of CCP in 100 ml of normalsaline, and mix adequately. The drug concentration is 1 g/ml.

Experimental process: Randomly divide 34 male Wister rats into fivegroups, and respectively administer the following doses of CCP: 5 g/kg,10 g/kg, 20 g/kg. Administer 10 ml/kg of normal saline of the samevolume to the control group, once a day for five consecutive days. Halfan hour after administering the drug on the fifth day, use ether toanaesthetize the rats. Using a total of 34 tubes, pour 0.1 ml of 3.13%sodium citrate in every tube to prevent the blood from coagulating.Administer a 0.1 g/kg aspirin once to the positive control group.Prepare platelet plasma according to the method explained previously. Dothe aggregation experiments, and record the conditions of every group.

Data processing: All data is shown with the mean±standard deviation(X±SD). All data between groups and within every group is examined usingthe t value in order to observe any significant difference. Experimentalresults are shown in Table 18.

TABLE 22 CCP's influence to platelet aggregation of rats (X ± SD) DoseMedicine (g/kg) Animal No. Aggregation ratio NS 0 7 65.33 ± 11.05 CCP5.0 7 56.19 ± 7.70 10.0 7 51.78 ± 9.68** 20 7 44.86 ± 7.99** Aspirin 0.16 26.58 ± 10.58** *P < 0.05, **P < 0.01, ***P < 0.001(compare withNormal saline)

The experiments about extracorporeal platelet aggregation of rabbitsmade it possible to find that by using 12.5 mg/ml of crude drug extractof CCP there is a significant reduction of the platelet aggregationratio (P<0.05). The higher the concentration (e.g., 37.5 mg/ml, 50mg/ml, 75 mg/ml) the more the platelet aggregation ratio drops. Inexperiments about extracorporeal platelet aggregation of rats, theextract of CCP can obviously reduce the platelet aggregation ratio whenthe dose ranges from 10 g/kg to 20 g/kg. The higher the dose, thestronger the effect. The results showed that the CCP could control therate of blood platelet aggregation induced by ADP.

Discussion

Adhesion, aggregation and exergic reactions of the platelet are thebasic functions under physiological conditions. It is also thethrombosis factor under pathological conditions.

An important method for the treatment of blood stasis is to control theplatelet aggregation and reduce blood viscosity in order to improveblood circulation. This is basically a method used to activate bloodcirculation in an attempt to dissipate blood stasis.

ADP results in blood aggregation by the ADP receptor. The resultsobtained from the experiments showed that the CCP could control theplatelet aggregation induced by ADP. The results also indicated theexistent of a dose-effect relationship.

After administering the CCP, there is a high level of control of theplatelet aggregation, which is induced by the ADP. This process showedthat the CCP has properties that activate blood circulation in order todissipate blood stasis.

Anti-Inflammation Effect

Material

Animal: Kunming mice, provided by the Experimental Animal Room in ourinstitute.

Medicines:

Extract of CCP, provided by Lianyungang Kanion Pharmaceutical Co. Ltd.,Batch No. 891011 (1 g extract corresponds to 4 g crude drug).

Bolus of Cinnamomi & Poria: produced by Shanxi Changzhi ChineseTradition Medicine Factory (1 g extract correspond to 4 g crude drug),Batch No. 900407

Hydrocortisone, produced by Harbin Third Pharmaceutical Factory, BatchNo. 900407

Instrument: Stiletto machine; Torsion balance

Methods

Preparation of drug: Put 12.5 g of extract of CCP in 100 ml of normalsaline, and mix adequately. The drug concentration is 0.5 mg/ml. Put 25g of extract of CCP in 100 ml of normal saline, and mix adequately. Thedrug concentration is 1 g/ml. Put 50 g of extract of CCP in 100 ml ofnormal saline, and mix adequately. The drug concentration is 2 g/ml. Put12.5 g of the Bolus of Cinnamomi & Poria in 100 ml of normal saline, andmix adequately. The drug concentration is 0.5 mg/ml. Put 25 g of extractin 100 ml of normal saline, and mix adequately. The drug concentrationis 1 g/ml. Put 50 g of extract in 100 ml of normal saline, and mixadequately. The drug concentration is 2 g/ml. Put 0.1 g ofHydrocortisone in 100 ml of normal saline. The drug concentration is 1mg/ml. Administer all three preparations (a total of seven differentsolutions) with 10 ml/kg.

Experimental method: According to their weight, randomly divide fiftymale mice into eight groups. Administer different doses of the sevensolutions mentioned above for three days and for the control groupadminister the normal saline of the same volume. After three days ofadministering the drug, besmear 0.5 ml croton oil on both sides of everymouse's left ear (compare with right ear). Two hours after administeringthe drug, kill the animals, and punch holes in the same location ontheir ears with 9 mm Stiletto machine, then weigh the ears of mice withTorsion balance.Swollen degree=left ear's weight−right ear's weight.

All data is shown with the mean±standard deviation (X±SD). Data betweengroups and within every group is examined using the t value in order toobserve any significant difference.

Results

Experimental results showed that a 10 g/kg CCP could reduce the degreeof ear swelling (p<0.01). It has been proven that an increase of thedose produces a reduction of the swelling. The degree of ear swelling ofthe group with a dosage of 20 g/kg of the extract of CCP is 5.92±3.11.Significant differences were found between the CCP in comparison withthe normal saline control group (p<0.001).

TABLE 23 Influence of the CCP on the ear swelling of mice Averageswelling Group Dose(g/Kg) Animal No. degree Control 0 10 18.36 ± 7.09CCP 5 10 17.75 ± 4.81 10 10  9.05 ± 5.92** 20 10  5.92 ± 3.11** BCP 5 1017.85 ± 3.06 10 10 10.90 ± 2.93 20 10  8.17 ± 5.91** Hydrocortisone0.025 10  4.68 ± 3.43***Discussion

Inflammatory agents can penetrate through the skin causing seriousdamage to the ear. Results from this experiment determined that a 10-20g/kg extract of CCP could reduce the swelling induced by an inflammatoryagent. The higher the doses of extract of the CCP, the stronger theeffect. The information reported from previous research validates thefact that the CCP has anti-inflammatory characteristics.

Conclusion

The extract of CCP can control the contraction frequency, the range andthe activity of the extra-corporeal uterine smooth muscle. It can alsoreduce rats' contraction frequency and activities of uterine smoothmuscle induced by oxytocin. The experiments proved that a 0.18 k/kgextract of CCP could decrease the twisting times of mice. By examiningthe Whole Blood Ratio Viscosity of rats, we found that a 5.0 g/kg ofcrude drug of CCP can reduce the Whole Blood Ratio Viscosity. Throughthis experiment is noticeable the existent difference with the normalsaline control group (low shear P<0.01, high shear P<0.05). Theexperiments indicated that the CCP could obviously reduce Whole BloodRatio Viscosity.

The results showed that a 12.5 mg/ml of crude drug of CCP could reducethe platelet aggregation rate. The platelet aggregation rate could bereduced if the CCP dose was increase (e.g., 37.5 mg/ml, 50 mg/ml, 75mg/ml). In regards to the results obtained from the experiment involvingthe platelet aggregation, it was proven that a 10 g/kg extract of CCPcould reduce the platelet aggregation ratio. The results indicated thata 10 g/kg extract of CCP could reduce auricular swelling (P<0.01)proving the strong anti-inflammatory effect of the CCP.

General Pharmacological Studies on Capsule of Cinnamomi and Poria (CCP)

According to the National guide principle for pre-clinical study of newdrug. Experiments were taken to assess the effect of Capsule ofCinnamomi and Poria (CCP) on the mental and nervous, cardiovascular andrespiratory systems of rats. The results indicated that there was nosignificant effects upon functions of those systems after the animalswere treated with CCP at doses of 250 mg/kg/day and 500 mg/kg/day for 5days.

Materials

Capsule of Cinnamomi and Poria (CCP) was provided by Lianyungang KanionPharmaceutical Co. Ltd. The batch number is 0002145 (the pan-mixed date:Mar. 30, 2000).

Wistar rats were provided by Nanjing Military Area Major HospitalExperiment Animal Center (NMAAMH-EAC), certificate number Su-Dong (Zhi)97001. Animals were raised in the animal house after purchased withtemperatures ranged from 18-24° C., light exposure was approximately 12hours. The food for animals was the granular rat food provided byNMAAMH-EAC The experiment begins after 2 weeks of breeding.

Equipments: JZ-1 tonotransducer, provided by Beijing Chenyungelectrotechnical Institue; CY-BK pressure transducer, provided by BeKoMeasure Control L.T. Co.; Ms 322 Multimedia Biology Signal AnalyticalSystem, provided by Guozhuo Pharmaceutical College.

Data were dealt with Pentium II 300 computer. The speed of record was0.2 s/cm and the plus were: ECG 1 mv/cm, BP 8 mv/cm, respiratory 8mv/cm, and all results are expressed as mean±S.D, and analysis bystudent t test.

Methods

30 adult rats were randomly divided into 3 groups, each contained 10rats, equal in both genders. CCP was given by gastric gavage to rats ata dose of 250 mg/kg or 500 mg/kg for 5 days. The other group was givensaline as control.

Observation for Nervous System: To observe the general appearance,behaviors, posture, gait of animals before and after the treatment. Payattention to signs like salivation, muscle trembling or changes of theeye pupil.

Observation for Cardiovascular system: The rats were under anesthesiabefore and after administration and were recorded for their BloodPressures (BP), Electrocardiogram (ECG) in II lead for the rates andrhythms of the heart.

Observation for Respiratory system: The animals were on anesthesia andtracheal cannular that were connect to the MBSAR to measure the changeof respiratory frequency.

Results:

The effects of CCP on Nervous system: The experiments showed that therewere no significant changes before and 1-56 hours after the treatmentfor such aspects as appearance, hairs, general behavior (appetite, andsleep), posture, and gait. Neither did CCP show effect on salivation,muscle trembling and change of the eye pupil.

The effect of CCP on cardiovascular and respiratory system: Theexperiment showed that there are no changes on Blood Pressure ECG, heartrate or respiratory frequencies between the control group andexperimental groups.

TABLE 24 The effect of CCP on heart rates of rats (Times/Min) Control250 g/kg 500 g/kg 322.8 ± 76.0 355.0 ± 67.3 387.9 ± 63.8

TABLE 25 The effect of CCP on BP of rats (Kpa) Control 250 g/kg 500 g/kg19.6 ± 4.2 21.2 ± 7.2 17.8 ± 6.1

TABLE 26 The effect of CCP on respiratory frequencies of rats (Time/Min)Control 250 g/kg BW 500 g/kg BW 83.1 ± 29.1 80.2 ± 18.3 76.2 ± 17.8Conclusion

This experiment showed that CCP has no effect on the mental, nervous,respiratory and cardiovascular systems in rats that were under thedosages of 250 mg/kg and 500 mg/kg during the 5 days experiment.

Acute Toxicity Study of CCP

20 mice of 18˜22 gram in body weight, equal in numbers of both genders,were given to CCP solution by gastric gavage 0.8 ml twice a day (246g/kg·day) for 7 days. Observe and make notes to the toxic effect,abnormal symptoms and deaths for 7 days. Animals were quirt and lessactive, but neither obvious reaction nor death could be observed whendrug dosage was raised up to 246 g/kg, a thousand times more thanhuman's dosage at 10 g a day.

Long-Term Toxicity Study of CCP

According to the National guide principle of pre-clinical study for anew drug. A chronic toxicity study on CCP is required. The result ofthis study were negative and it indicated that there was not any reverseeffect acting on the functional and structural internal organs of therats after treated with CCT at doses of 250 mg/kg/d, 500 mg/kg/d and1000 mg/kg/d for a 3 months period.

Materials

Capsule of Cinnamomi and Poria (CCP) was provided by Lianyungang KanionPharmaceutical Co. Ltd. The batch number is 0002145 (the pan-mixed date:Mar. 30, 2000).

Wistar rats were provided by Nanjing Military Area Major HospitalExperiment Animal Center (NMAAMH-EAC), certificate number Su-Dong (Zhi)97001. Animals were raised in the animal house after purchased withtemperatures ranged from 18-24° C., light exposure was approximately 12hours. The food for animals was the granular rat food provided byNMAAMH-EAC The experiment begins after 2 weeks of breeding.

Method

The adult rats were divided into 4 groups randomly, each group contained20 animals (10 males and 10 females). The rats was given CCP by gastricgavage at doses of 250 mg/kg/d, 500 mg/kg/d, and 1000 mg/kg/d, 6 days aweek for 3 months. The other group was given saline as a control group.

Parameters Evaluated

General Appearances, behaviors, excretions, appetite, and weightchanges. After 24 hours of the last dose, the rats were sacrificed andwere observed for the following index.

1) Hematology: The count of RBC, WBC, platelet and hemochrome.

2) Biochemical parameters of blood: AST, ALT, BUN, TP, ALB, GLU, T-BIL,Crea, and T-CHO.

3) Histopathology examination: The important organs such as pituitary,heart, liver, kidney, lung, testis, ovary, uterus, lymph, stomach,thyroid, duodenum, ileum, and colon were all dissected out and fixed inBounin's solution. Then the organs were to be embedded in paraffin waxand serially sectioned. The sections were stained with H. & E. stain andexamined by a light microscope.Results

There were no changes or any difference between the control group andCCP groups on rat's appearances, behaviors, excretions, appetite, bodyweight and weight of the organs. The rats that were given CCP by gastricgavage at dose of 250 mg/kg/d, 500 mg/kg/d, and 1000 mg/kg/d for a 3months period did not showed any treatment related signs of effect.

TABLE 27 Effect of CCP on important organs weight Heart Lung KidneyLiver Control 0.39 ± 0.04 0.78 ± 0.02 0.95 ± 0.07 4.82 ± 0.26 250 mg0.37 ± 0.05 0.86 ± 0.07 1.06 ± 0.08 4.92 ± 0.52 500 mg 0.38 ± 0.03 0.79± 0.04 1.06 ± 0.08 4.80 ± 0.36 1000 mg  0.45 ± 0.03 0.80 ± 0.05 1.04 ±0.10 4.70 ± 0.39 Spleen Uterus Ovary Control 0.18 ± 0.01 0.17 ± 0.030.028 ± 0.004 250 mg 0.16 ± 0.01 0.17 ± 0.02 0.022 ± 0.008 500 mg 0.18 ±0.01 0.17 ± 0.01 0.028 ± 0.004 1000 mg  0.19 ± 0.02 0.17 ± 0.03 0.028 ±0.003

There were no significant change between the control and CCP groups incount of RBC, WBC, platelet and hemochrome during 3 months of theexperiment. The CCP groups were given the dosages of 250 mg/kg/day, 500mg/kg/day or 1000 mg/kg/day.

TABLE 28 The effect of CCP on hematology parameters RBC WBC PlateletGroup 10⁶/mm³ 10³/mm³ 10³/mm³ Hb g/100 ml Control 4.49 ± 0.53 15.2 ± 4.61170 ± 141 13.1 ± 1.6 250 mg/kg 3.95 ± 0.41 13.6 ± 4.1 1159 ± 156 12.2 ±1.1 500 mg/kg 4.56 ± 0.51 15.6 ± 5.0 1073 ± 76 13.0 ± 2.0 1000 mg/kg 4.28 ± 0.39 14.7 ± 4.5 1129 ± 137 12.4 ± 2.0

There was no significant change or difference between the CCP groups andthe control group in parameters of AST, ALT, BUN, Crea, TP, GLU, T_BIL,and T-CHO. It was suggested that CCP has no effects on the function ofliver, kidney, and the metabolism of protein, Carbohydrates, and fatduring the 3 months of the experiment. The dosages of CCP that weregiven to the rats were 250 mg/kg/d, 500 mg/kg/d, and 1000 mg/kg/d.

TABLE 29 The effect of CCP on the functions of the liver and kidney. BUNCr Group AST (u) ALT (u) (mg/100 ml) (mg/100 ml) Control 18.3 ± 8.6 13.8± 2.7 39.7 ± 7.6 1.38 ± 0.23 250 mg/kg 18.3 ± 4.2 15.3 ± 5.1 15.3 ± 5.11.32 ± 0.25 500 mg/kg 15.4 ± 6.7 17.2 ± 6.0 39.1 ± 8.4 1.38 ± 0.23 1000mg/kg  17.3 ± 6.6 15.3 ± 4.3 38.6 ± 10.0 1.41 ± 0.21

TABLE 30 The effect of CCP on the metabolism of Carbohydrates, proteinand fat TP ALB GLU T-BIL T-CHO Group g/100 ml g/100 ml mg/100 ml mg/100ml mg/100 ml Control 5.96 ± 1.08 4.09 ± 0.82 101.6 ± 12.1 0.37 ± 0.18113.1 ± 16.9 250 mg/kg 6.28 ± 0.92 4.39 ± 0.86 114.3 ± 12.2 0.39 ± 0.19117.5 ± 19.9 500 mg/kg 6.35 ± 0.86 4.08 ± 0.87  99.8 ± 13.3 0.38 ± 0.18127.3 ± 28.6 1000 mg/kg  6.30 ± 0.87 4.22 ± 0.57  99.0 ± 15.4 99.0 ±15.4 120.5 ± 21.4

Histopathology examinations: Many of the organs such as pituitary,heart, liver, kidney, lung, testis, ovary, uterus, lymph, stomach,duodenum, ileum, and colon were all dissected and were put into Bounin'ssolution. Then the organs were to be embedded in paraffin wax andserially sectioned. The sections were stained with H.& E. stain andexamined under microscope. The results of all three treated groupsshowed no structural and other changes that were compared to the controlgroup.

Conclusion

The results indicated that there were no significant effects on thefunctions and structure of all examined organs, during the 3 monthsperiod of experiment that the rats were given the dose of CCP by 250mg/kg/d, 500 mg/kg/d and 1000 mg/kg/d. In conclusion, the experimentshowed that CCP are safe for usage.

Effect on Genital System of Female Young Rat

Materials

Capsule of Cinnamomi and Poria (CCP) was provided by Lianyungang KanionPharmaceutical Co. Ltd. (Batch No. 990515)

Radio-immunoassay Kits for oestadiol (Sensitivity: 4 pg/ml; variationindex between batches: 5.0, within batch: 4.0) and pregnendione(Sensitivity: 0.2 ng/ml; variation index between batches: 7.4, withinbatch: 5.2), provided by Jiuding Biomedical company Ltd., Tianjin

30 female Wistar rats, 4 weeks of age, provided by Nanjing Military AreaMajor Hospital Experiment Animal Center (NMAAMH-EAC), certificate numberSu-Dong (Zhi) 97001. Animals were raised in the animal house of ourinstitute, the temperatures was ranged from 18-24° C., light exposurewas approximately 12 hours. Animal's food was the standard granular ratfood provided by NMAAMH-EAC.

Method

30 rats were randomly divided into high, low doses and control groups,10 rats in each group. The three groups of rats were administered withCCP 1,500 mg/kg·day, 300 mg/kg·day, or normal saline 0.4 ml/rat for 7days. Animals were killed on the eighth day, uteruses and ovaries wereweighted immediately and blood sample toke from the abdominal aorta forconcentration of serum oestadiol and pregnendione.

Result

Two animals, one in the high dose and the other the control group diedone hour after the gastric infusion. We considered that the death wascaused by inappropriate operation of gastric infusion since certainbloody fluid was found in the mouths of both rats. No other abnormalcases were found during the test for their growth, body developingaction and reactions.

TABLE 31 Weight of genital organs after administrations Mean ± SD BodyWeight of Weight Index of Number weight uterus Index of of ovary ovaryGroup of rat (g) (mg) uterus (mg) (mg) Control 9 63.8 ± 3.5 114.8 ± 18.3180.6 ± 31.9 54.8 ± 5.0 86.3 ± 10.1 High dose 9 66.7 ± 6.7 130.4 ± 12.3196.2 ± 13.7 58.4 ± 5.7 88.1 ± 8.7 Low dose 10 68.9 ± 6.0 120.5 ± 22.0177.6 ± 34.0 55.2 ± 7.6 80.4 ± 10.3

In compare with the control, there was no significant difference in theCCP groups (P>0.05)

TABLE 32 Level of Serum oestadiol and pregnendione. OestadiolPregnendione Group Number of rat (pg/ml) (ng/mg) Control 9 4.1 ± 1.40.47 ± 0.22 High dose 9 4.3 ± 2.1 0.60 ± 0.45 Low dose 10 3.6 ± 1.1 0.63± 0.41

In compare with the control, there was no significant difference in theCCP groups (P>0.05)

Conclusion

CCP showed no obvious adverse effect on the genital systems of youngadult rat (4 weeks) after 7 days administration of high/low doses.

The Micronucleus Test of CCP

According to the Chinese National Guide Principle of pre-clinical studyfor new drugs. A Micronucleus Test was conducted. By the results of thistest, we would be able to tell the mutagenesis effect of CCP and therange of the effect. It was found that there was no increasingmicronucleus after treated with CCT by gastric gavage at doses of 2.5g/kg/day, 1.25 g/kg/day and 0.625 g/kg/day for 2 days.

Materials

ICR adult mouse were provided by Nanking Military Area Major HospitalExperimental Animal Center, certificate number: Su-dong (Zhi) 95040.Animals were raised in the animal house after purchased withtemperatures ranged from 18-24° C., light exposure was approximately 12hours. The food for animals was the granular rat food provided byNMAAMH-EAC The experiment begins after 1 weeks of breeding.

Capsule of Cinnamomi and Poria (CCP) was provided by Lianyungang KanionPharmaceutical Co. Ltd. The batch number is 0002145 (the pan-mixed date:Mar. 30, 2000).

Method:

30 mice were randomly divided into 5 groups, each group contained 6animals. Three of the groups were drug-groups and animals in them weregiven to CCP by gastric gavage at doses of 2.5 g/kg/day, 1.25 g/kg/dayand 0.625 g/kg/day for 2 days. (LD50 of CCP from an acute toxicity testwas 5 g/Kg, we adopted a half dose for our study). The fourth group isgiven to normal saline (0.5 ml/mice) as a negative control group. Thelast group was given to cyclophosphamide (CP) at 40 mg/Kg via abdominalcavity injection as a positive control group.

Animals were killed 24 hours after administration. Both femurs of themice were taken for sampling. Femurs were pressed to move out themarrow, with a drop of calf serum added to it, smear the marrow on aplate. After the smeared plate was fixed in the solution of methyl for10 minutes, it was dyed with the Giemsa's method and observed under theoil lens of a microscope. In the principle of double-blinded study, PCEand MNPCE cells were counted. 1000 PCE and MNPCE cell were counted foreach mice, calculated the ratio of micronuclei over the 1000 cells. Theratio of micronuclei represents the dose-effectiveness relation. Thedata was analyzed with statistical t test.

Results

It was found that the incidence of micronucleus in the group ofcyclophosphamide injection was obviously higher than the negativecontrol. Yet, there was no statistical difference between CCP groups andthe negative control for the incidence of micronucleus in mice's marrowcells.

TABLE 33 The number of micronuclei in mice administered with differentdosages of CCP with the control groups Group Animals PCE CellsMicronuclei Negative control 6 6000 12 Positive control 6 6000 381 2.500g/Kg 6 6000 25 1.250 g/Kg 6 6000 18 0.625 6 6000 17

TABLE 34 The incidence of micronuclei in mice administered withdifferent dosages of CCP with the control groups Group X ± SD RangeNegative control  2.6777 ± 1.0328 1-4 Positive control 63.5000 ± 20.637343-98 2.500 g/Kg  401667 ± 2.4833 2-9 1.250 g/Kg  3.0000 ± 1.7889 1-60.625 g/Kg  2.8333 ± 2.1370 1-7Conclusion

High (2.500 g/Kg), middle (1.250 g/Kg), and low (0.625 g/Kg) dosages ofCCP had no significant effect on improving the incidence of micronucleusin mice bone marrow cells.

Therefore, we have concluded that this drug had no harmful effect onchromatosome.

The Ames Test of CCP

In compliance with the Chinese National Guide Principle of pre-clinicalstudy for new drugs, an “Ames” test on CCP was performed to decide thepossible effect of mutagenesis of the drug. It was found that afterblending of CCP into bacterial strains TA97, TA98, TA100 and TA102 atconcentrations of 5 mg/plate, 0.5 mg/plate, and 0.05 mg/plate, thenumber of back mutation colonies in each culture plate did not increasewith or without the ‘S9’. Meanwhile, the positive control showed anincreased number of back mutation colonies. This test demonstrated thatCCP had no mutagenesis effect.

In this experiment, the blending of the suspend drug in different dosesinto the culture plates of S. Typhimurium mutational strains withoutHistidine might cause changes in the number of back mutation colonies ifthe drug is effective in mutagenensis.

Materials

S. Typhimurium TA97, TA98, TA100, and TA102 were provided by EpidemicPrevention Station, Jiangsu, People's Republic of China. The bacterialstrains were cultured at 37° C. in vibrate water bath until the numberof bacteria raised up to 1×10⁹; CCP was provided by the LianyungangKanion Pharmaceutical Co. Ltd., batch number: 0002145 (the pan mixeddate: Mar. 30, 2000).

Drugs for positive control: 1) solution of sodium azide, add 1.5 mlsodium azide (1.5 ug/1.0 ml) into 500 ml water. Filter the solution toeliminate contaminating bacteria in it. 2) solution of 2-amino fluorine,add 40 ml 2-amino fluorine (200 ug/1.0 ml) into 200 ml water, Filter thesolusion to eliminate contaminating bacteria in it.

Hepatic microsome enzyme (S9): provided by Jiangsu Epidemic PreventionStation, People's Republic of China.

Reagents: glucose-6-phosphate, coenzyme II, histidine, and biotin wereprovided by Shanghai Biochemistry Reagent Company, the agar by JapanNAGASKITG Biochemistry Company. The rest of the reagents were producedby Nanjing Chemistry Reagent Company, People's Republic of China.

Methods:

1) The first step of the test was to establish a sketch on its bacterialinhibition effect and the dosages used for the second step test inrelation to the possible effect: a) Bacillus coli were inoculated into aconical flask of nutrient broth medium (about 20 ml), and put intoincubator at 37° C. for 24 hours for the increase of bacteria number. b)The test was operated on 9 cm bacteria culture plates. Add 5.0 mg, 0.5mg, 0.05 mg of CCP, or 0.1 ml of normal saline into plates with 15 ml ofnutrient agar and 0.1 ml the bacteria fluid. The plates were cultured inincubator at 37° C. for 24 hours. After incubation, the number ofcolonies in each plate had no significant difference. The result showedthat CCP had no bacterial inhibition effect even with the enhancedexperimental dosages.2) Step two: a) Inoculated S. Typhimurium TA97, TA98, TA100, and TA102respectively into conical flasks with nutrient's broth medium (about 20ml) and cultivated them in incubator at 37° C. for 24 hours. b) The testwas operated in 9 mm culture plates following regular procedures for thepreparation of reagents, culture plates and suspending drugs ofdifferent dosages, the inoculation of bacteria strains, cultivation inincubator etc. c) Three suspending drug groups of low, middle, and highdosages, one negative control (auto reverse) and two positive controls(sodium azide and 2-amino fluorine) were setup for the test. Each of theabove mentioned groups were operated for three cultural platesobservation. Concentrations of drugs and agents of each group wereshowed in table 31

TABLE 35 The groups for Ames test TA97, TA98, Normal sodium 2-amino −S9TA100 or TA102 Saline CCP azide fluorine +S9 (buffer, Groups (ml/plate)(ml/plate) (ml/plate) (μg/plate) (μg/plate) (ml/plate) ml/plate)Negative 0.1 1.0 0.5 0.5 Ctrl Positive 0.1 1.5 0.5 (Sodium axide)Positive 0.1 10.0 0.5 0.5 (2-amino fluorine) CCP low 0.1 0.05 0.5 0.5CCP mid 0.1 0.50 0.5 0.5 CCP high 0.1 5.00 0.5 0.5Results:

The number of Back Mutation Colonies (BMC) in each CCP dose group wasnot increased with or without the presence of S9. The positive controlgroups had been increased significantly for the back mutation colonies.

TABLE 36 The Number of Back Mutation Colony in Ames Test Back MutationColony (colony/plate, mean ± SD) TA 97 TA 98 TA 100 TA 102 Group −S9 +S9−S9 +S9 −S9 +S9 −S9 +S9 Negative 147.7 ± 2.1 152.0 ± 29.5 24.7 ± 3.224.0 ± 3.66  99.3 ± 8.4 117.1 ± 14.6 186.0 ± 39.8 215.7 ± 33.0 ControlSodium 1120.0 ± 233.5** 1072.3 ± 215.9** 1163.0 ± 92.3**   1005.7 ±8301** axide 2-amino 1109.7 ± 58.8**  1131.7 ± 233.2** 1095.7 ± 118.2**1080.0 ± 89.4** fluorine CCP low 156.7 ± 12.2 169.3 ± 12.1 22.0 ± 1.027.3 ± 4.5 101.7 ± 9.0 106.0 ± 18.1 236.0 ± 73.4 258.0 ± 71.3 CCP mid 84.7 ± 7.5 118.3 ± 12.3 24.7 ± 4.5 25.0 ± 6.2 150.3 ± 6.7 149.3 ± 12.7238.0 ± 64.2 233.3 ± 43.9 CCP high 127.0 ± 20.0 146.0 ± 29.1 26.7 ± 4.028.0 ± 1.7 146.3 ± 12.7 156.3 ± 18.0 247.7 ± 21.4 218.7 ± 19.7 **Incompare with the negative control (auto reverse), the two positivegroups showed the most significant difference (P < 0.001).Conclusion

The test showed that CCP had no mutagenesis effect on the 4 strains ofHistidine Defect S. Typhimurium.

Evaluation of CCP for Carcinogenicity in Cellular MalignancyTransformation on Mammal's Cells In Vitro

According to the National guide principle of pre-clinical study for newdrugs. The carcinogensis of CCP was studied with cellular malignancytransformation test. Study on the Syrian Hamster embryo cell in vitro,the results indicated that there was no malignant cell clonetransference found among 50 culture flasks tested with CCP at dosagesfrom 10 mg/flask to 0.625 mg/flask. The malignant transformation testconcluded that there was no carcinogensis effect of CCP

Materials

Capsule of Cinnamomi and Poria (batch number: 0002145, TCM) was providedby Lianyungang Kanion Pharmaceutical Co. Ltd.; The Syrian Hamster embryo(SHE) cell was donation from Dr. Li Xiangming, Medical College ofYangZhou University; RPMI 1640 medium and culture flasks were productsof Gibco Co.; Penicillin and streptomycin were products from Yananpharmaceutical factory, Shanghai; Fetal bovine serum was from Jinlingbiotechnology Co., Nanjing; The rest of chemicals or reagents were fromJiangsu Chemical Reagent Co.

CO₂ Incubator was purchased from Lab-Line instruments Inc., USA. YJ-875clean worktable was from the Suzhou cleaning Equipment Factory.Multifunction microscope was purchased from Olympus Co. Japan. CO₆₀radiative was provided by Academy of Agricultural Science Jiangsu,China.

Method

Cultivation of the SHE cell: In sterile circumstance, the ample of SHEcell was unfrozen and restored in a water bathe at 37° C. and inoculatedinto several culture flasks. Cultured at 37° C., humid, with 5% CO₂ and95% air, the cell was to be washed in Hank's balanced salt solution(BBS) after a 24 hours culturing in order to remove the dimethylsulfoxide (DMSO). The cells were to be cultured in the RPMI medium 1640with 10% fetal calf serum, penicillin and streptomycin for the other 24hours. After that, the cells were seeded at 1×10⁵ viable cells per 5 mlculture medium in 30 ml flasks and cultured for 48 hours. After the 48hours, the cells were exposed to CO₆₀ radiation for a dosage of 70 Gy.Cells were prepared as “nourishing cell” after the above procedures.

On the third day of the experiment, another ample of SHE cell wasrestored and cultured for proliferation, but were not exposed to CO60radiation. They were what we called the “target cell”. The target cellswere inoculated at 1×10³ per flask to the nourishing cell's flasks onthe sixth day of cultivation and the seeded flasks were to be culturedfor another 1 or 2 days.

CCP was grinded into a fine powder substance and was blended withculture medium. The prepared drug medium was then filled into the flasksof cells and the cells were exposed to CCP drug medium and cultivatedfor 24 hours before the drug medium was removed and the cells werewashed 3 times in the BBS to remove the remaining drugs. The flasks ofcells were then filled with “drug-free” medium and were cultivated for10-12 days. During the days, the culture medium was scheduled to changedonce every other day.

Result

A total 70 flasks of 7 groups were prepared, 10 flasks for each group.Group A, B, C, D, and E were exposed to the test drug—CCP at dosages of10 mg/flask, 5 mg/flask, 2.5 mg/flask, 1.25 mg/flask, and 0.625mg/flask. Group F was a positive control with MNNG at 5 mg/flask. GroupG was negative control. After the final culture, the cells were takenout and chemically fixed in methanol for 15 minutes. Dried in the air,the cells were dyed in 10% Giemsa for 20 minutes and then, they wereready to be studied under the microscope. The cells were observed fortheir appearance and colonies morphologically in cell formation ortransformation.

There are differences between normal and malignant transformed cellcolony in cell morphology in visions under a phase contrast microscope.The cells and colonies were observed and recorded for 61-70 cultureflasks (9 cultures were discarded due to bacterial contamination). Thecolony forming efficiency (CE) was calculated as:

${CE} = {\frac{{Number}\mspace{14mu}{of}\mspace{14mu}{Cell}\mspace{14mu}{inoculated}}{{Number}\mspace{14mu}{of}\mspace{14mu}{Colony}\mspace{14mu}{Grown}} \times 100\%}$

The results were listed in table 33.

TABLE 37 Colony Forming Efficiency (CE) in groups of cells Normal cellMalignant transformed Group Number colony (CE) cell colony (CE) A: 10.0mg/flask 9 22.6 ± 11.8 0 B: 5.0 mg/flask 8 27.9 ± 13.7 0 C: 2.5 mg/flask7 17.0 ± 7.8 0 D: 1.25 mg/flask 10 20.6 ± 9.2 0 E: 0.625 mg/flask 9 12.6± 6.7 00 Positive Control 8  2.6 ± 4.5 4.4 ± 3.7 Negative control 1019.4 ± 12.6 0Conclusion

There was no malignant transformed cell colony that was discovered inthe groups of CCP. The criteria for the malignant cells determination weadopted was that of the Dunkle criteria (1981). As a conclusion, CCP isof no positive carcinogensis effect upon Syrian Hamster embryo cell.

Clinical Trial of CCP

Total amount of cases: 150 (100 cases in the CCP group, 50 cases in thecontrol groups).

Both from outpatient and inpatient departments in the following threeunits:

-   -   Department of Gynecology, Hospital of TMC, Jiangsu, People's        Republic of China.    -   Department of Gynecology, Hospital of TMC, Nanjing, People's        Republic of China.    -   Department of TCM, Obstetrical and Gynecological Hospital,        Nanjing, Jiangsu, People's Republic of China.

Patients age: 20˜54, max 63, min 20, average 37; Course of illness: max17 years, min 1 month, average 25.59 months (Refer to Tables 38 and 39for distribution of age and courses). All the 150 cases were randomlygrouped as: 100 cases in the CCP drug group and 50 cases in the controlgroup.

There was a match-pair grouping within the 100 cases of CCP drug group.Among the 100 patients, 50 cases were chosen for the match-pair group(A), which included cases of intramural hysteromyoma and that of chronicpelvic inflammations with inflammatory lower abdomen masses. The other50 patients were designed to be in the non-pair group (C), whichincluded diseases of dysmenorrheas, dysfunctional uterine bleedingcaused by irregular shedding of endometrim, hemotcele of uterine cavity,remote extrauterine pregnancy, and postpartum lochiorrhea atc. Thecontrol group was categorized as (B).

The match-pair group (A) was intended to be as similar as possible togroup B regarding severity of diseases, courses, ages, etc.

Diagnoses Based on Symptoms in TCM

Symptom Class A:

-   -   Profuse menstruation    -   Non-stop Metrostaxis    -   Excess of blood clots during menstrual period    -   Inflammatory lower abdomen mass (es)    -   Vague pain, distending pain or press refusal at lower abdomen    -   Dim purpuric spots in tongue (TCM index)    -   Faint wrist pulse (TCM index)        Symptom Class B:    -   Lumber pain    -   Large amount of leulorrhea    -   Heavy and distending anus    -   Symptoms get worse during menstruation or periods of tiredness        Symptom of Lab Tests (Class C)    -   Abnormal indexes in blood rheology    -   Increased blood platelet aggregation, or enhanced release of        blood platelet        Diagnosis in Western Traditional Medicine        1) Small Leiomyoma uteri    -   Lower abdomen mass(es)    -   Profuse or prolonged menstruation, excess of blood clots during        menstrual period, vague pain, distending pain at lower abdomen,        or anemia.    -   Enlarged uterine, but no larger than a pregnant uterine of 2        months (rigid or ultra sound proved).        2) Chronic Pelvic Inflammations with Inflammatory Lower Abdomen        Masses

Symptoms: vague pain, distending pain, heavy or distending lowerabdomen, and lumber pains. Symptoms get worse during menstruation orperiods of tiredness. Other symptoms include slightly high temperature,heavy and distending anus, and large amount of leulorrhea.

Signs: mostly posterior uterine or fixed uterine. Perceivable ropeshaped rigid mass at one side of uterine that could generate pain whenthe area is pressed. Masses or thicker tissues can be felt in one orboth sides of the uterine. B-ultrasound proved.

3) Dysfunctional Uterine Bleeding Caused by Irregular Shedding ofEndometrim.

Symptoms: prolonged period of menstruation in a normal month circle(more than seven days). Large amounts of blood during menstruation withblack blots or distending pain at lower abdomen.

Clinical signs: normal circle with two-phase body temperature curve.Temperature would not fall to its level of follicular phase after thebeginning of menstruation. Physical exam or B ultrasound demonstratedthat there were no organic changes

Criteria of TCM for Enlistment of Patients:

1) More than 3 items of class A,

2) Or, one item of class A plus two items class C

3) Or, one item of class A plus two items of class B and one item ofclass C

4) Anyone's diagnosis in the three western traditional medicines

Exclusion of Cases:

1) Patients with other organic diseases, abdomen masses in women withpostmenopausal period or found to be malignant.

2) Interruption of treatment, uncompleted data record.

3) Patients were screened by blood routine test that included bloodplatelets counting, bleeding and coagulating time, hepatic and kidneyfunction tests. The abnormal menstruation caused by blood, hepatic orkidney function was eliminated.

Method:

Capsule of Cinnamomi and Poria, three capsules each administration,three times a day. Control group will take Bolus of Cinnamomi and Poriaone bolus each time, three times a day for 3 months. For patients ofintramural hysteromyoma, stop the administration during menstruation.For patients of dysfunctional uterine bleeding, stop the administrationright after bleeding.

Lab Tests:

Except for the routine blood tests, all patients were required to takethe following:

Aggregation ratio of blood platelet for 1 and 5 minutes

Ratio of whole blood viscosity

Ratio of plasma viscosity

Erythrocyte sedimentation

Packed blood cell volume

Reduced whole blood viscosity

K value of sedimentation

Points for Judgment of Effectiveness:

For small intramural hysteromyoma: enlarged uterine, irregular shape ofuterine, large amount of menstruation and excess of black clots (thepatient got five points for each of the above). Non-stopped Metrostaxis,vague pain in lower abdomen, distending pain or press refusal (thepatient got two points).

For chronic pelvic inflammations with inflammatory lower abdomen masses:confined or fixed uterine and/or tenderness, rigid or rope shapedoviduct and/or tenderness. Thickened tissue in one or both side ofuterine and/or tenderness, mass in one or both side of pelvic cavity(the patient got five points for each). For aching pains in lowerabdomen or lumber, the patient got three points. For large amounts ofleulorrhea, and if symptoms worsen during menstruation or periods oftiredness, the patient got two points.

For dysfunctional uterine bleeding caused by irregular shedding ofendometrim: prolonged menstruation for more than seven days, largeamount of menstruation, excess of blood blots, luteal phase for 12-14days or it would not fall to its level of follicular phase after thestarting of menstruation (the patient got five points for each of thesesymptoms). For pain in the lower abdomen, the patient got two points.

For all diagnosis, if dim purpuric spots in tongue or faint wrist pulsewere observed, add 1 point for each. Half a point is given for eachadditional year of illness if the diagnosis is small intramuralhysteromyoma or chronic pelvic inflammations with inflammatory lowerabdomen masses. Half a point for every three months if the diagnosis isdysfunctional uterine bleeding caused by irregular shedding ofendometrim.

Degree of Severity:

Serious: >20 points

Medium: 10˜20 points

Slight: <10 points

Definition and Evaluation of Effectiveness:

1) Rehabilitation: recovered from any symptoms, abnormal physical signsand lab tests. Total point=0 and no recurrence within 6 month aftertreatment.

2) Obvious effect: revered from any symptoms, abnormal physical signs.Index of lab test improved obviously, at least ⅔ lower the total pointsthan that of before treatment

3) Improved: some of the symptoms, abnormal physical signs and lab testsimproved. A ⅓ reduction in the total amount of points when compared tothe amount obtained before the treatment.

4) No effect: no improvement.

Results

1) See Table 40 for comparison of points and table 41 for improvementrate between groups. Table 40 and 41 shows the improvement of thesymptoms. They show both CCP and BCP groups are getting better thanthose before treatment. No significant different between these 2 groups2) See table 5 for changes of abnormal physical signs3) Table 43 shows changes of lab tests (blood rheology, platelets etc.)of patient groups before and after treatment.

CCP groups A: rehabilitation 14 (28%), obvious effect 16, andimprovement 18. Rehabilitation plus obvious effect 60%, total effectiverate 96%.

BCP group (control): R 12, OE 17, and IM 15. R rate 24%, R+OE rate 58%,total off rate 88%, X² test demonstrate that no significant differencebetween the 2 groups (P>0.05)

CCP A+C group (100 cases): R rate 27%, R+OE rate 58%, total off rate 96%

Five patients (three cases in CCP, 3% and two in BCP, 4% group)complained of a slight pain or discomfort in their upper abdomen aftertaking the drugs. They all recovered in two to three hours after theystopped using the drug or took the advice of using the drug only aftermeals.

Discussion

In TCM diagnosis system, the above mentioned multiple diagnosis: primaryor secondary dysmenorrhea, dysfunctional uterine bleeding caused byirregular shedding of endometrium, chronic pelvic inflammations withinflammatory lower abdomen masses or small intramural hysteromyoma areunder the same title of diagnostic catalog in TCM and are considered toshare the same etiological cause. They were studied and treated the sameway with the same drugs.

The title is “Diseases of Blood Stasis” in OB/GYN. The common age ofsuch diseases is 25˜54 (women of childbearing age). The course of suchdiseases ranges from 1 month to 17 years.

Both CCP and BCP may reduce the symptoms of such diseases, especiallyfor the following symptoms in Class A and B (improvement of more than80%) profuse menstruation, non-stopped Metrostaxis, excess of bloodclots during menstrual period, vague pain or distending pain lowerabdomen, aching pain at lumber, heavy and distending anus. For othersymptoms in Class A and B, there was a 70% improvement. Effect of CCPand BCP on above symptoms showed no significant difference (table 37)

Abdomen masses detected by physical examinations or B-ultrasound inpatients with inflammatory lower abdomen masses or small intramuralhysteromyoma: in CCP groups the physical exam showed 28% of masses haddisappeared, 36% shrank, 44% were controlled in size, and 2% enlarged.On B-ultrasound screen 30% disappeared, 36% shrank, 32% were controlledin size, and 2% enlarged. In BCP, similar results were found. There wasno significant difference between the two groups (P>0.05) (table 42)

Total points: both CCP and BCP groups showed differences before andafter the treatment (P<0.001). No significant differences were foundbetween the two groups in any single diagnosis (P>0.05) except that inthe matched pairs analysis, the point fall in CCP was larger than thatof BCP (P≈0.05) table 43.

In observation of the laboratory study made with the different groups,the six indexes of blood rheology study and the two indexes for plateletaggregation were carefully recorded and analyzed. The CCP group showedsignificant differences after treatment in each of the eight indexeswhile BCP changes were noticeable in six out of the eight (exceptaggregation ratio of platelet at one minute and erythrocytesedimentation). The results show that CCP or BCP could change thecondition of the blood flow and circulation. For unknown reasons,comparison between match pair groups A and B showed that there were somesignificant differences between the two groups except for the ratio ofwhole blood viscosity and reduced whole blood viscosity. It wasindicated that CCP had stronger effects on blood flow.

In laboratory studies, CCP showed positive results in improving typicalsymptoms and complains, physical signs and examinations, and blood flowindexes. The capsule appears to be as effective as BCP which is asuccessful TCM preparation in treating the above-mentioned diseases andhad been practiced for decades in China. The CCP even showed morebenefits than the BCP.

TABLE 38 Distribution of disease duration in the groups Number Max MinGroup Dignosis of Case (month) (month) X S t _(X1− Xt) A LU + IM 50 1001 25.92 34.3908 0.1308 B LU + AIM 50 204 1 24.98 37.3928 C DUB etc. 50156 1 25.86 33.0972 A + C Mixed cases 100 100 1 25.89 33.9720 A LU 25100 2 29.92 37.5488 0.1598 IM 25 120 1 21.92 31.1688 0.0341 B LU 25 2042 20.36 40.0145 IM 25 160 1 21.60 35.0678 LU: Leiomyoma Uteri; IM:Inflammatory Mass; DUB: Dysfunctional Uterine Bleeding

TABLE 39 Changes in points before and after treatment Before AfterSubtract of treatment treatment points Group Diag. case X S X S d Sd tαt _(X1− Xt) A LU + IM 50 21.5 6.3374 5.11 4.9315 16.39 6.0426 19.17961.9123 25-34 35-44 45-54 Group yr. (%) yr. (%) yr. (%) Total X² A 16(32) 26 (52)  8 (16) 50 0.000 B 16 (32) 26 (52)  8 (16) 50 C 27 (54) 13(26) 10 (20) 50 A + C 43 (43) 39 (39) 18 (18) 100 B LU + IM 50 20.727.5958 6.77 6.3465 13.95 6.7000 14.7225 C DUB 50 17.96 7.7800 6.105.8379 11.86 6.6078 12.6914 A + C Mixed cases 100 19.73 7.2803 5.6055.3994 14.125 6.6982 21.0878 A LU 25 24.06 4.9986 6.44 5.5945 17.626.6274 13.2933 1.7981 IM 25 18.94 6.5849 3.78 3.8299 15.16 5.241514.4615 0.83514 B LU 25 22.34 7.5605 8.22 6.7628 14.12 7.1272 9.9057 IM25 19.10 7.4274 5.32 5.6677 13.78 6.3870 10.7876

TABLE 40 Changes in Clinical symptoms Cases Cases observed improved Rateof before After improvement Symptoms Group treatment treatment (%) X²Profuse A 29 27 93.10 0.668 menstruation B 28 24 85.71 C 40 39 97.50 A +C 69 66 95.65 Non-stopped A 21 18 85.71 0.013 Metrostaxis B 19 16 84.21C 20 18 90.00 A + C 41 36 87.8 Lots of blood A 33 31 93.94 0.059 clotsduring B 34 30 88.24 menstrual C 44 42 95.45 period A + C 77 73 94.81Vague pain at A 47 45 95.74 1.14 lower abdomen B 48 43 89.58 C 35 3497.14 A + C 82 79 96.34 Dim purpuric A 32 25 78.13 0.154 spots in B 3324 72.73 tongue C 31 27 87.10 A + C 63 52 82.54 Faint wrist A 34 2779.41 0.155 pulse B 31 23 74.19 C 33 27 81.82 A + C 67 54 80.60 Achingpain A 36 32 38.89 1.249 at lumber B 30 23 76.67 C 34 31 91.18 A + C 7060 90 Large amount A 25 22 88.0 0.003 of leulorrhea B 24 21 87.50 C 1915 78.95 A + C 43 36 83.72 Heavy and A 17 16 74.12 0.257 distending B 1816 83.89 anus C 15 14 93.33 A + C 32 30 93.75 Symptoms get A 18 14 77.780.022 worse during B 16 12 75 menstruation C 20 17 85 or exhaustion A +C 38 31 81.58

TABLE 41 Changes of abdomen masses in physical examination andB-ultrasound Vanished Shrunk No change Enlarged Mean Group Cases (%) (%)(%) (%) grade Physical A 50 14 (28) 13 (26) 22 (41) 1 (2) 2.80 0.1443exam B 50  9 (18) 22 (44) 16 (32) 3 (6) 2.74 B- A 50 15 (30) 18 (36) 16(32) 1 (2) 2.96 0.2337 ultra B 50  8 (16) 22 (44) 12 (24) 3 (6) 2.50sound

TABLE 42 Changes in indexes in blood rheolgy before After Subtract titem Grp X1 S1 X2 S2 d − S d t d d1 − d2 Ratio A 1.532 0.167 1.460 0.2190.0724 0.0293 2.469 1.2856 of B 1.591 0.150 1.512 0.192 0.079 0.02143.688 whole C 1.577 0.286 1.502 0.282 0.026 0.0250 1.023 blood A + C1.554 0.235 1.531 0.261 0.023 0.0116 1.989 viscosity Ratio A 1.585 0.1081.530 0.129 0.055 0.0176 3.104 8.1715 of B 1.620 0.120 1.536 0.127 0.0840.0184 4.571 plasma C 1.577 0.285 1.527 0.128 0.050 0.040 1.250viscosity A + C 1.597 0.124 1.531 0.127 0.066 0.0128 5.140 Erythrocyte A20.860 14.777 24.620 10.825 4.240 1.721 2.463 13.0023 sedimentation C25.160 11.467 23.760 10.3205 1.400 1.035 1.352 A + C 27.010 13.02724.190 10.583 2.820 1.009 2.7940 Packed A 40.940 6.769 39.480 5.73651.460 1.050 1.391 3.4771 blood B 43.327 4.233 41.135 4.530 2.202 1.0842.031 cell C 41.600 5.341 40.380 4.060 1.220 0.719 1.698 volume A + C41.270 6.106 39.930 4.990 1.340 0.633 2.117 Reduced A 10.1022 2.7209.604 2.392 0.499 0.293 1.700 0.785 whole B 9.849 2.717 9.307 2.3800.542 0.259 2.094 blood C 9.865 3.002 9.556 2.665 0.309 0.501 0.616viscosity A + C 9.984 2.871 9.580 2.523 0.404 0.203 1.991 K A 93.40843.377 78.809 36.608 14.599 4.726 3.089 7.383 value B 97.815 47.71089.168 34.789 8.647 3.188 2.712 of C 86.106 36.955 78.599 33.239 7.5122.937 1.861 sedimentation A + C 89.757 40.461 78.704 34.912 11.053 3.1123.552 Changes in aggregation of platelet Before After Subtract t itemgroup N X1 S₁ X2 S₂ d − S d t d d1 − d2 1′ A 42 42.647 20.874 35.21016.431 7.437 2.300 3.233 11.864 B 44 39.620 16.379 40.872 17.313 1.2442.529 0.492 C 48 45.613 21.483 43.030 15.960 2.581 3.025 0.854 A + C 9044.130 21.127 39.120 16.212 5.010 2.103 2.382 5′ A 42 65.475 26.32553.245 24.062 12.232 3.978 3.075 5.245 B 44 56.256 25.758 48.092 26.3288.164 3.188 2.561 C 48 63.490 25.715 63.637 21.951 0.148 0.148 0.118 A +C 90 64.420 26.015 58.440 23.480 5.980 2.981 2.006

TABLE 43 Comparison of drug effectiveness between groups Comprehensiveanalysis Ratio of rehabil- Obvious im- No Rehabil- Group itation effectproved effect n itation X² A 14 16 18 2 50 14 (24.0) 0.2079 B 12 17 15 650 12 (24.0) C 13 15 18 4 50 13 (26.0) A + C 27 31 36 6 100 26 (27.0)0.1559 Ratio of Total obvious effective group effect X² rate (%) X² μ A30 (60.0) 0.0413 48 (96.0) 2.1739 0.2887 B 29 (58.0) 44 (88.0) C 28(56.0) 46 (92.0) A + C 58 (58.0) 0.2 94 (94.0) 1.6304

REFERENCES

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1. A method for treating small intramural hysteromyoma in a subjectsuffering therefrom, comprising administering to the subject aneffective amount of a composition comprising extracts from the stem ofCinnamomum cassia, sclerotium of Poria cocos, root bark of Paeoniasuffruticosa, root of Paeonia lactiflora and seed of Prunus persica orPrunus davidiana, wherein the composition comprises 1.3-1.9% ofPaeoniflorin and 0.7-1.1% of Paeonol.
 2. The method of claim 1, whereinthe stem of Cinnamomum cassia, sclerotium of Poria cocos, root bark ofPaeonia suffruticosa, root of Paeonia lactiflora and seed of Prunuspersica or Prunus davidiana are obtained from cultivated plants.
 3. Themethod of claim 1, wherein the composition further comprises apharmaceutically acceptable carrier.
 4. The method of claim 3, whereinthe composition is formulated in the form of a pill, capsule, granule,tablet, suspension, injection, syrup or tincture.